Molecules with extended half-lives, compositions and uses thereof

ABSTRACT

The present invention provides molecules, including IgGs, non-IgG immunoglobulins, proteins and non-protein agents, that have increased in vivo half-lives due to the presence of an IgG constant domain, or a portion thereof that binds the FcRn, having one or more amino acid modifications that increase the affinity of the constant domain or fragment for FcRn. Such proteins and molecules with increased half-lives have the advantage that smaller amounts and or less frequent dosing is required in the therapeutic, prophylactic or diagnostic use of such molecules.

This application is a continuation of U.S. Ser. No. 11/649,455, filedJan. 3, 2007, which is a continuation of U.S. Ser. No. 11/397,328, filedApr. 3, 2006, which is a continuation of U.S. Ser. No. 10/020,354 (nowU.S. Pat. No. 7,083,784), filed Dec. 12, 2001, which claims the benefitof U.S. provisional application Ser. Nos. 60/254,884, filed Dec. 12,2000, and 60/289,760, filed May 9, 2001, and each of which isincorporated by reference herein in its entirety.

This invention was made, in part, with United States Government supportunder award number A139167 from the National Institute of Health. TheUnited States Government may have certain rights in the invention.

1. INTRODUCTION

The present invention relates to molecules whose in vivo half-lives areincreased by modification of an IgG constant domain, or FcRn (FcReceptor-neonate) binding domain thereof. Specifically, these moleculeshave amino acid modifications that increase the affinity of the constantdomain or fragment thereof for the FcRn. Increasing the half-life oftherapeutic and diagnostic IgGs and other bioactive molecules usingmethods of the invention has many benefits including reducing the amountand/or frequency of dosing of these molecules, for example, in vaccines,passive immunotherapy and other therapeutic and prophylactic methods.The invention further relates to fusion proteins containing all or aportion (a FcRn binding portion) of an IgG constant domain having one ormore of these amino acid modifications and a non-IgG protein ornon-protein molecule conjugated to such a modified IgG constant domain,where the presence of the modified IgG constant domain increases the invivo half-life of the non-IgG protein or molecule.

2. BACKGROUND OF THE INVENTION

The use of immunoglobulins as therapeutic agents has increaseddramatically in recent years and have expanded to different areas ofmedical treatments. Such uses include treatment of agammaglobulinemiaand hypogammaglobulinemia, as immunosuppressive agents for treatingautoimmune diseases and graft-vs.-host (GVH) diseases, the treatment oflymphoid malignancies, and passive immunotherapies for the treatment ofvarious systemic and infectious diseases. Also, immunoglobulins areuseful as in vivo diagnostic tools, for example, in diagnostic imagingprocedures.

One critical issue in these therapies is the persistence ofimmunoglobulins in the circulation. The rate of immunoglobulin clearancedirectly affects the amount and frequency of dosage of theimmunoglobulin. Increased dosage and frequency of dosage may causeadverse effects in the patient and also increase medical costs.

IgG is the most prevalent immunoglobulin class in humans and othermammals and is utilized in various types of immunotherapies anddiagnostic procedures. The mechanism of IgG catabolism in thecirculation has been elucidated through studies related to the transferof passive immunity from mother to fetus/neonate through the placenta oryolk sac or through colostrum (maternofetal transfer of IgG viatranscytosis) in rodents (Brambell, Lancet, ii:1087-1093, 1966;Rodewald, J. Cell Biol., 71:666-670, 1976; Morris et al., In: AntigenAbsorption by the Gut, pp. 3-22, 1978, University Park Press, Baltimore;Jones et al., J. Clin. Invest., 51:2916-2927, 1972).

The involvement of certain receptors in the maternofetal transmission ofmaternal IgGs was first suggested by Brambell's group in their study onthe intestinal absorption of maternal antibodies from ingested milk innewborn rats (Halliday, Proc. R. Soc. B., 143:408-413, 1955; Halliday,Proc. R. Soc. B., 144:427-430, 1955; Halliday, Proc. R. Soc. B.,148:92-103, 1957; Morris, Proc. R. Soc. B., 148:84-91, 1957; Brambell etal., Proc. R. Soc. B., 149:1-11, 1958; Morris, Proc. R. Soc. B.,160:276-292, 1964). Brambell et al. suggested, based on the observationthat heterologous IgGs interfered with the transmission of a specificantibody, that IgG molecules from various species might havesufficiently similar structures or sequences that bind to commonreceptors (Brambell et al., Proc. R. Soc. B., 149:1-11, 1958).

A high-affinity Fc receptor, FcRn, has been implicated in this transfermechanism. The FcRn receptor has been isolated from duodenal epithelialbrush borders of suckling rats (Rodewald et al., J. Cell Biol.,99:154s-164s, 1984; Simister et al., Eur. J. Immunol., 15:733-738, 1985)and the corresponding gene has been cloned (Simister et al., Nature,337:184, 1989 and Cold Spring Harbor Symp. Quant. Biol., LIV, 571-580,1989). The later clonings of FcRn-encoding genes from mice (Ahouse etal., J. Immunol., 151:6076-6088, 1993) and humans (Story et al., J. Exp.Med., 180:2377-2381, 1994) demonstrate high homology of these sequencesto the rat FcRn, suggesting a similar mechanism of maternofetaltransmission of IgGs involving FcRn in these species.

Meanwhile, a mechanism for IgG catabolism was also proposed byBrambell's group (Brambell et al., Nature, 203:1352-1355, 1964;Brambell, Lancet, ii:1087-1093, 1966). They proposed that a proportionof IgG molecules in the circulation are bound by certain cellularreceptors (i.e., FcRn), which are saturable, whereby the IgGs areprotected from degradation and eventually recycled into the circulation;on the other hand, IgGs which are not bound by the receptors aredegraded. The proposed mechanism was consistent with the IgG catabolismobserved in hypergammaglobulinemic or hypogammaglobulinemic patients.Furthermore, based on his studies as well as others (see, e.g.,Spiegelberg et al., J. Exp. Med., 121:323-338, 1965; Edelman et al.,Proc. Natl. Acad. Sci. USA, 63:78-85, 1969), Brambell also suggestedthat the mechanisms involved in maternofetal transfer of IgG andcatabolism of IgG may be either the same or, at least, very closelyrelated (Brambell, Lancet, ii:1087-1093, 1966). Indeed, it was laterreported that a mutation in the Fc-hinge fragment caused concomitantchanges in catabolism, maternofetal transfer, neonatal transcytosis,and, particularly, binding to FcRn (Ghetie et al., Immunology Today,18(12):592-598, 1997).

These observations suggested that portions of the IgG constant domaincontrol IgG metabolism, including the rate of IgG degradation in theserum through interactions with FcRn. Indeed, increased binding affinityfor FcRn increased the serum half-life of the molecule (Kim et al., Eur.J. Immunol., 24:2429-2434, 1994; Popov et al., Mol. Immunol.,33:493-502, 1996; Ghetie et al., Eur. J. Immunol., 26:690-696, 1996;Junghans et al., Proc. Natl. Acad. Sci. USA, 93:5512-5516, 1996; Israelet al., Immunol., 89:573-578, 1996).

Various site-specific mutagenesis experiments in the Fc region of mouseIgGs have led to identification of certain critical amino acid residuesinvolved in the interaction between IgG and FcRn (Kim et al., Eur. J.Immunol., 24:2429-2434, 1994; Medesan et al., Eur. J. Immunol., 26:2533,1996; Medesan et al., J. Immunol., 158:2211-2217, 1997). These studiesand sequence comparison studies found that isoleucine at position 253,histidine at position 310, and histidine at position 435 (according toKabat numbering, Kabat et al., In: Sequences of Proteins ofImmunological Interest, US Department of Health and Human Services,1991, which is hereby incorporated by reference in its entirety), arehighly conserved in human and rodent IgGs, suggesting their importancein IgG-FcRn binding.

Additionally, various publications describe methods for obtainingphysiologically active molecules whose half-lives are modified either byintroducing an FcRn-binding polypeptide into the molecules (WO 97/43316;U.S. Pat. No. 5,869,046; U.S. Pat. No. 5,747,035; WO 96/32478; WO91/14438) or by fusing the molecules with antibodies whose FcRn-bindingaffinities are preserved but affinities for other Fc receptors have beengreatly reduced (WO 99/43713) or fusing with FcRn binding domains ofantibodies (WO 00/09560; U.S. Pat. No. 4,703,039). However, none ofthese publications disclose specific mutants in the IgG constant domainthat affect half-life.

Prior studies have demonstrated that certain constant domain mutationsactually reduce binding to FcRn and, thereby, reduce the IgG in vivohalf-life. PCT publication WO 93/22332 (by Ward et al.) disclosesvarious recombinant mouse IgGs whose in vivo half-lives are reduced bymutations between about residue 253 and about residue 434. Particularly,substitutions of isoleucine at position 253; histidine at position 310;glutamine at position 311; His at position 433; and asparagine atposition 434 were found to reduce IgG half-life.

Modulation of IgG molecules by amino acid substitution, addition, ordeletion to increase or reduce affinity for FcRn is also disclosed in WO98/23289; however, the publication does not list any specific mutantsthat exhibit either longer or shorter in vivo half-lives.

In fact, only one mutant of mouse IgG1 that actually exhibited increasedhalf-life, the triple mutation Thr252 to Ala, Thr254 to Ser, and Thr256to Phe, has been identified (WO 97/34631).

In view of the pharmaceutical importance of increasing the in vivohalf-lives of immunoglobulins and other bioactive molecules, there is aneed to develop modified IgGs and FcRn-binding fragments thereof,(particularly modified human IgGs) that confer increased in vivohalf-life on immunoglobulins and other bioactive molecules.

3. SUMMARY OF THE INVENTION

The present invention is based upon the inventors' identification ofseveral mutations in the constant domain of a human IgG molecule thatincrease the affinity of the IgG molecule for the FcRn. In particular,the present inventors have screened libraries of human IgG1 constantdomains with random amino acid mutations introduced into particularregions of the constant domain for increased affinity for FcRn. Suchrandom mutations were made in the regions of residues 251-256, 285-290,and 308-314, all of which are in CH2 domain, and 385-389 and 428-436,which are in CH3 domain, of human IgG1 hinge-Fc regions (residues asdepicted in FIG. 2 (SEQ ID NO:83 or analogous residues in hinge-Fcregions of other IgG molecules as determined by sequence alignment). Asused herein, all residues of the IgG constant domain are numberedaccording to Kabat et al. (Sequences of Proteins of ImmunologicalInterest, U.S. Department of Health and Human Services, 1991, which isincorporated by reference herein in its entirety) and as presented inFIG. 2 (SEQ ID NO:83), and include corresponding residues in other IgGconstant domains as determined by sequence alignment. The in vivohalf-life, or persistence in serum or other tissues of a subject, ofantibodies, and other therapeutic agents and other bioactive moleculesis an important clinical parameter which determines the amount andfrequency of antibody (or any other pharmaceutical molecule)administration. Accordingly, such molecules, including antibodies, withincreased half-life are of significant pharmaceutical importance.

Thus, the present invention relates to a modified molecule (preferably aprotein, but may be a non-protein agent) that has an increased in vivohalf-life by virtue of the presence of a modified IgG constant domain,or FcRn-binding portion thereof (preferably the Fc or hinge-Fc domain)(preferably from a human IgG) wherein the IgG constant domain, orfragment thereof, is modified (e.g., by amino acid substitution,deletion or insertion) to increase the affinity for the FcRn. In aparticular embodiment, the present invention relates to modified IgGs,whose in vivo half-lives are extended by the modification of amino acidresidues identified to be involved in the interaction of the hinge-Fcdomain with the FcRn receptor. Preferably, the constant domain orfragment thereof has higher affinity for FcRn at pH 6.0 than at pH 7.4.Such modifications may also alter (i.e., increase or decrease) thebioavailability (e.g., transport to mucosal surfaces, or other targettissues) of the molecules. The invention also relates to other types ofimmunoglobulins or fragments thereof (i.e., non-IgG immunoglobulins),non-immunoglobulin proteins and non-protein agents that are fused orconjugated to, or engineered to contain, an IgG constant domain, orFcRn-binding fragment thereof, having one or more such amino acidmodifications.

In preferred embodiments, the present invention provides molecules,particularly, immunoglobulins whose in vivo half-lives are extended bythe presence of an IgG constant domain, or FcRn binding fragment thereof(preferably, Fc or hinge-Fc domain), that has modifications of one ormore of amino acid residues 251-256, 285-290, 308-314, 385-389, and428-436 that increase the affinity of the constant domains or fragmentsthereof for FcRn. In certain embodiments, these modifications preferablyexclude residues 252, 254, and 256, in particular when the IgG constantdomain or fragment thereof, is murine. In particular embodiments, themodification is at one or more surface-exposed residues, and themodification is a substitution with a residue of similar charge,polarity or hydrophobicity to the residue being substituted. Inpreferred embodiments, the modified IgG constant domain, or fragmentthereof, binds with higher affinity to FcRn at pH 6.0 than at pH 7.4. Ina preferred embodiment, the constant domain, or fragment thereof, ismodified by substitution of one or more of amino acid residues 251-256,285-290, 308-314, 385-389, and 428-436 that increase the affinity of theconstant domain or FcRn-binding fragments thereof for FcRn. In certainembodiments, substitutions of residue 252 with leucine, residue 254 withserine, and/or residue 256 with phenylalanine are excluded, particularlywhen the constant domain or fragment thereof is derived from a mouseIgG.

In specific embodiments, the invention provides immunoglobulins or otherbioactive molecules that contain an IgG1 constant domain, orFcRn-binding fragment thereof (preferably Fc or hinge-Fc domain)(preferably human), having amino acid modifications at one or more ofposition 308, 309, 311, 312, and 314, more specifically, havingsubstitutions at one or more of positions 308, 309, 311, 312 and 314with threonine, proline, serine, aspartic acid and leucine respectively.In another embodiment, residues at one or more of positions 308, 309,and 311 are substituted with isoleucine, proline, and glutamic acid,respectively. In yet another embodiment, residues at one or more ofpositions 308, 309, 311, 312, and 314, are substituted with threonine,proline, serine, aspartic acid, and leucine, respectively. The inventionfurther relates to combinations of these amino acid substitutions.

Furthermore, the invention provides immunoglobulins or other bioactivemolecules that contain an IgG1 constant domain, or FcRn-binding fragmentthereof (preferably, Fc or hinge-Fc domain) (preferably human), havingamino acid modifications at one or more of positions 251, 252, 254, 255,and 256, more specifically, having substitutions at one or more of thesepositions. In specific embodiments, residue 251 is substituted withleucine or arginine, residue 252 is substituted with tyrosine,phenylalanine, serine, tryptophan or threonine, residue 254 issubstituted with threonine or serine, residue 255 is substituted withleucine, glycine, isoleucine or arginine, and/or residue 256 issubstituted with serine, arginine, glutamine, glutamic acid, asparticacid, alanine, asparagine or threonine. In a more specific embodiment,residue 251 is substituted with leucine, residue 252 is substituted withtyrosine, residue 254 is substituted with threonine or serine, and/orresidue 255 is substituted with arginine. In yet another specificembodiment, residue 252 is substituted with phenylalanine and/or residue256 is substituted with aspartic acid. In a preferred embodiment,residue 251 is substituted with leucine, residue 252 is substituted withtyrosine, residue 254 is substituted with threonine or serine, and/orresidue 255 is substituted with arginine. The invention further relatesto any combination of these substitutions.

Furthermore, the invention provides immunoglobulins or other bioactivemolecules that contain an IgG1 constant domain, or FcRn-binding fragmentthereof (preferably, Fc or hinge-Fc domain) (preferably human), havingamino acid modifications at one or more of positions 428, 433, 434, and436, more specifically, having substitutions at one or more of thesepositions. In specific embodiments, residue 428 is substituted withmethionine, threonine, leucine, phenylalanine, or serine, residue 433 issubstituted with lysine, arginine, serine, isoleucine, proline,glutamine, or histidine, residue 434 is substituted with phenylalanine,tyrosine, or histidine, and/or residue 436 is substituted withhistidine, asparagine, arginine, threonine, lysine, methionine, orthreonine. In a more specific embodiment, residues at one or morepositions 433, 434, and 436 are substituted with lysine, phenylalanine,and histidine, respectively. In a preferred embodiment, residue 428 issubstituted with methionine and/or residue 434 is substituted withtyrosine.

Furthermore, the invention provides immunoglobulins or other bioactivemolecules that contain an IgG1 constant domain, or FcRn-binding fragmentthereof (preferably, Fc or hinge-Fc domain) (preferably human), havingamino acid modifications at one or more positions 385, 386, 387, and389, more specifically, having substitutions at one or more of thesepositions. In specific embodiments, residue 385 is substituted witharginine, aspartic acid, serine, threonine, histidine, lysine, oralanine, residue 386 is substituted with threonine, proline, asparticacid, serine, lysine, arginine, isoleucine, or methionine, residue 387is substituted with arginine, histidine, serine, threonine, alanine, orproline and/or residue 389 is substituted with proline or serine. Inmore specific embodiments, residues at one or more positions 385, 386,387, and 389 are substituted with arginine, threonine, arginine, andproline, respectively. In yet another specific embodiment, residues atone or more positions 385, 386, and 389 are substituted with asparticacid, proline, and serine, respectively.

Molecules of the invention include any combination of theabove-described substitutions at one or more of residues 251, 252, 254,255, 256, 308, 309, 311, 312, 385, 386, 387, 389, 428, 433, 434, and/or436. In a preferred embodiment, the molecule of the invention contains aFc region, or FcRn-binding domain thereof, having one or more of thefollowing substitutions: leucine at residue 251, tyrosine at residue252, threonine or serine at residue 254, arginine at residue 255,threonine at residue 308, proline at residue 309, serine at residue 311,aspartic acid at residue 312, leucine at residue 314, arginine atresidue 385, threonine at residue 386, arginine at residue 387, prolineat residue 389, methionine at residue 428, and/or tyrosine at residue434.

Included within the invention are pharmaceutical compositions andmethods of prophylaxis and therapy using modified immunoglobulins,proteins and other bioactive molecules of the invention having extendedhalf-lives. Also included are methods of diagnosis using modifiedimmunoglobulins, proteins and other bioactive molecules of the inventionhaving extended half-lives. In a specific embodiment, the inventionprovides an anti-respiratory syncytial virus (RSV) antibody useful totreat or prevent RSV infection, such as SYNAGIS® (see U.S. Pat. No.5,824,307 and Johnson et al., J. Infectious Disease 176:1215-1224, 1997,both of which are incorporated by reference in their entireties), andother anti-RSV antibodies, including variants of SYNAGIS® (see U.S.patent application Ser. No. 09/724,396, filed Nov. 28, 2000, U.S. patentapplication Ser. No. 09/724,531, filed Nov. 28, 2000, U.S. patentapplication Ser. No. 09/996,288, filed Nov. 28, 2001 (attorney docketno. 10271-047), and U.S. patent application Ser. No. 09/996,265, filedNov. 28, 2001 (attorney docket no. 10271-048), all entitled “Methods ofAdministering/Dosing Anti-RSV Antibodies for Prophylaxis and Treatment,”all by Young et al., all of which are incorporated by reference hereinin their entireties, particularly the sequences of heavy and light chainvariable domains and CDRs of anti-RSV antibodies disclosed therein),which has one or more amino acid modifications in the constant domainthat increase the affinity of the antibody for FcRn and that has anincreased in vivo half-life (see also, Section 5.1 infra).

3.1 Definitions

The term “IgG Fc region” as used herein refers the portion of an IgGmolecule that correlates to a crystallizable fragment obtained by papaindigestion of an IgG molecule. The Fc region consists of the C-terminalhalf of the two heavy chains of an IgG molecule that are linked bydisulfide bonds. It has no antigen binding activity but contains thecarbohydrate moiety and the binding sites for complement and Fcreceptors, including the FcRn receptor (see below). The Fc fragmentcontains the entire second constant domain CH2 (residues 231-340 ofhuman IgG1, according to the Kabat numbering system) (e.g., SEQ IDNO:80) and the third constant domain CH3 (residues 341-447) (e.g., SEQID NO:81).

The term “IgG hinge-Fc region” or “hinge-Fc fragment” as used hereinrefers to a region of an IgG molecule consisting of the Fc region(residues 231-447) and a hinge region (residues 216-230; e.g., SEQ IDNO:82) extending from the N-terminus of the Fc region. An example of theamino acid sequence of the human IgG1 hinge-Fc region is SEQ ID NO:83.

The term “constant domain” refers to the portion of an immunoglobulinmolecule having a more conserved amino acid sequence relative to theother portion of the immunoglobulin, the variable domain, which containsthe antigen binding site. The constant domain contains the CH1, CH2 andCH3 domains of the heavy chain and the CHL domain of the light chain.

The term “FcRn receptor” or “FcRn” as used herein refers to an Fcreceptor (“n” indicates neonatal) which is known to be involved intransfer of maternal IgGs to a fetus through the human or primateplacenta, or yolk sac (rabbits) and to a neonate from the colostrumthrough the small intestine. It is also known that FcRn is involved inthe maintenance of constant serum IgG levels by binding the IgGmolecules and recycling them into the serum. The binding of FcRn to IgGmolecules is strictly pH-dependent with optimum binding at pH 6.0. FcRncomprises a heterodimer of two polypeptides, whose molecular weights areapproximately 50 kD and 15 kD, respectively. The extracellular domainsof the 50 kD polypeptide are related to major histocompatibility complex(MHC) class I α-chains and the 15 kD polypeptide was shown to be thenon-polymorphic β₂-microglobulin (β₂-m). In addition to placenta andneonatal intestine, FcRn is also expressed in various tissues acrossspecies as well as various types of endothelial cell lines. It is alsoexpressed in human adult vascular endothelium, muscle vasculature andhepatic sinusoids and it is suggested that the endothelial cells may bemost responsible for the maintenance of serum IgG levels in humans andmice. The amino acid sequences of human FcRn and murine FcRn areindicated by SEQ ID NO:84 and SEQ ID NO:85, respectively. Homologs ofthese sequences having FcRn activity are also included.

The term “in vivo half-life” as used herein refers to a biologicalhalf-life of a particular type of IgG molecule or its fragmentscontaining FcRn-binding sites in the circulation of a given animal andis represented by a time required for half the quantity administered inthe animal to be cleared from the circulation and/or other tissues inthe animal. When a clearance curve of a given IgG is constructed as afunction of time, the curve is usually biphasic with a rapid α-phasewhich represents an equilibration of the injected IgG molecules betweenthe intra- and extra-vascular space and which is, in part, determined bythe size of molecules, and a longer β-phase which represents thecatabolism of the IgG molecules in the intravascular space. The term “invivo half-life” practically corresponds to the half life of the IgGmolecules in the β-phase.

An “isolated” or “purified” antibody or fusion protein is substantiallyfree of cellular material or other contaminating proteins from the cellor tissue source from which the protein is derived, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized. The language “substantially free of cellular material”includes preparations of an antibody or a fusion protein in which theantibody or the fusion protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. Thus, anantibody or a fusion protein that is substantially free of cellularmaterial includes preparations of antibody or fusion protein having lessthan about 30%, 20%, 10%, or 5% (by dry weight) of contaminatingprotein. When the antibody or the fusion protein is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, 10%, or 5% of thevolume of the protein preparation. When the antibody or the fusionprotein is produced by chemical synthesis, it is preferablysubstantially free of chemical precursors or other chemicals, i.e., itis separated from chemical precursors or other chemicals which areinvolved in the synthesis of the protein. Accordingly such preparationsof the antibody or the fusion protein have less than about 30%, 20%,10%, 5% (by dry weight) of chemical precursors or compounds other thanthe antibody or antibody fragment of interest. In a preferred embodimentof the present invention, antibodies are isolated or purified. Inanother preferred embodiment of the invention, fusion proteins areisolated or purified.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. An “isolated” nucleic acid molecule does notinclude cDNA molecules within a cDNA library. In a preferred embodimentof the invention, nucleic acid molecules encoding antibodies areisolated or purified. In another preferred embodiment of the invention,nucleic acid molecules encoding fusion proteins are isolated orpurified.

The term “host cell” as used herein refers to the particular subjectcell transfected with a nucleic acid molecule or infected with phagemidor bacteriophage and the progeny or potential progeny of such a cell.Progeny of such a cell may not be identical to the parent celltransfected with the nucleic acid molecule due to mutations orenvironmental influences that may occur in succeeding generations orintegration of the nucleic acid molecule into the host cell genome.

The names of amino acids referred to herein are abbreviated either withthree-letter or one-letter symbols.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoacid or nucleic acid sequence). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=numberof identical overlapping positions/total number of positions×100%). Inone embodiment, the two sequences are the same length.

The determination of percent identity between two sequences can also beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl.Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul,1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al.,1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performedwith the NBLAST nucleotide program parameters set, e.g., for score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid molecules of the present invention. BLAST protein searches can beperformed with the XBLAST program parameters set, e.g., to score-50,wordlength=3 to obtain amino acid sequences homologous to a proteinmolecule of the present invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively,PSI-BLAST can be used to perform an iterated search which detectsdistant relationships between molecules (Id.). When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g.,http://www.ncbi.nlm.nih.gov). Another preferred, non-limiting example ofa mathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithmis incorporated in the ALIGN program (version 2.0) which is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

4. DESCRIPTION OF THE FIGURES

FIG. 1 shows the structure of the IgG hinge-Fc region indicating thelocations of the residues identified to be involved in the interactionwith the FcRn receptor (Ghetie et al., Immunology Today, 18(12):592-598,1997).

FIG. 2 shows the amino acid sequence of the human IgG1 hinge-Fc region(SEQ ID NO:83) containing a hinge region (SEQ ID NO:82), CH2 domain (SEQID NO:80), and CH3 domain (SEQ ID NO:81).

FIGS. 3 (A and B) show the amino acid sequences of (A) human FcRn (SEQID NO:84) and (B) mouse FcRn (SEQ ID NO:85), respectively.

FIG. 4 shows the amino acid sequence of the human IgG1 hinge-Fc region(SEQ ID NO:83), in which wild-type residues which are mutated by aminoacid substitutions are indicated in underlined bold-face.

FIG. 5 shows a schematic diagram of panning process for thephage-displayed modified hinge-Fc library.

FIG. 6 shows a summary of the occurrence of selected mutant residues atthe variant positions in the libraries screened.

FIGS. 7 (A-D). (A) shows the binding of murine FcRn to immmobilized IgG1having M252Y/S254T/T256E substitutions. Murine FcRn was injected at 10different concentrations ranging from 1 nM to 556 nM over a surface onwhich 4000 resonance units (RU) of IgG1 had been coupled. Afterequilibrium was reached, residual bound protein was eluted with a pulseof PBS, pH 7.4. (B) shows the binding of human FcRn to immmobilizedIgG1/M252Y/S254T/T256E. Murine FcRn was injected at 8 differentconcentrations ranging from 71 nM to 2.86 μM over a surface on which1000 RU of IgG1 had been coupled. After equilibrium was reached,residual bound protein was eluted with a pulse of PBS, pH 7.4. (C) and(D) show scatchard analyses of the data in (A) and (B), respectively,after correction for nonspecific binding. R_(eq) is the correctedequilibrium response at a given concentration C. The plots are linearwith correlation coefficients of 0.97 and 0.998, respectively. Theapparent K_(d) are 24 nM and 225 nM, respectively.

FIGS. 8 (A-H). (A)-(D) show the results from BIAcore analysis of thebinding of murine FcRn at pH 6.0 and pH 7.4 to (A) wild type human IgG1,(B) M252Y/S254T/T256E, (C) H433K/N434F/Y436H, and (D) G385D/G386P/N389S,respectively, after correction for nonspecific binding. Murine FcRn wasinjected at a concentration of 1.1 μm over a surface on which 1000 RU ofwild type IgG1, 1000 RU of M252Y/S254T/T256E, 955 RU ofH433K/N434F/Y436H, and 939 RU of G385D/Q386P/N389S had been coupled.(E)-(H) show the results from BIAcore analysis of the binding of humanFcRn at pH 6.0 and pH 7.4 to (E) wild type human IgG1, (F)M252Y/S254T/T256E, (G) H433K/N434F/Y436H, and (H) G385D/Q386P/N389S,respectively, after correction for nonspecific binding. Human FcRn wasinjected at a concentration of 1.4 μm over a surface on which 1000 RU ofwild type IgG1, 1000 RU of M252Y/S254T/T256E, 955 RU ofH433K/N434F/Y436H, and 939 RU of G385D/Q386P/N389S had been coupled.

FIG. 9 shows the space-filling model of the surface of the Fc fragmentof a human IgG1 based upon the human IgG1 structure of Deisenhofer,1981, Biochemistry 20:2361-2370. Residues are color-coded according tothe gain of free energy of stabilization of the Fc-FcRn complex: red,substitutions at these positions were found to increase affinity b afactor of at least 2.5 times in the Fc/human FcRn interaction and of atleast 5 time in the Fc/mouse FcRn interaction; blue, substitutions atthose positions were found to increase affinity by a factor of less than2 times in both the Fc-human FcRn and Fc-mouse FcRn interaction. Thefigure was drawn using Swiss pdb viewer (Guex and Peitsch, 1997,Electrophoresis 18:2714-2723).

FIG. 10 shows the changes in serum concentration ([Mab] ng/ml) over time(in days) of antibody having a wild type constant domain (SYNAGIS®)(open squares), or constant domains with the following mutations:M252Y/S254T/T256E (open circles), G385D/Q386P/N389S (solid squares), andH433K/N434F/Y436H (solid circles). Antibody concentration was determinedusing anti-human IgG ELISA.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to molecules, particularly proteins, moreparticularly immunoglobulins, that have an increased in vivo half-lifeand comprise an IgG constant domain, or fragment thereof that binds toan FcRn (preferably a Fc or hinge-Fc domain), that contains one or moreamino acid modifications relative to a wild type IgG constant domainwhich modifications increase the affinity of the IgG constant domain, orfragment thereof, for the FcRn. In a preferred embodiment, the inventionparticularly relates to the modification of human or humanized IgGs andother bioactive molecules containing FcRn-binding portions of humanIgGs, which have particular use in human therapy, prophylaxis anddiagnosis.

5.1 Molecules with Increased In Vivo Half-Lives

The present invention is based upon identification of amino acidmodifications in particular portions of the IgG constant domain thatinteract with the FcRn, which modifications increase the affinity of theIgG, or fragment thereof, for the FcRn. Accordingly, the inventionrelates to molecules, preferably proteins, more preferablyimmunoglobulins, that comprise an IgG constant domain, or FcRn bindingfragment thereof (preferably a Fc or hinge-Fc domain fragment), havingone or more amino acid modifications (i.e., substitutions, insertions ordeletions) in one or more regions that interact with the FcRn, whichmodifications increase the affinity of the IgG or fragment thereof, forthe FcRn, and also increase the in vivo half-life of the molecule. Inpreferred embodiments, the one or more amino acid modifications are madein one or more of residues 251-256, 285-290, 308-314, 385-389, and428-436 of the IgG hinge-Fc region (for example, as in the human IgG1hinge-Fc region depicted in FIG. 4, SEQ ID NO:83), or analogous residuesthereof, as determined by amino acid sequence alignment, in other IgGhinge-Fc regions. In a preferred embodiment, the amino acidmodifications are made in a human IgG constant domain, or FcRn-bindingdomain thereof. In a certain embodiment, the modifications are not madeat residues 252, 254, or 256 (i.e., all are made at one or more ofresidues 251, 253, 255, 285-290, 308-314, 385-389, or 428-436) of theIgG constant domain. In a more preferred embodiment, the amino acidmodifications are not the substitution with leucine at residue 252, withserine at 254, and/or with phenylalanine at position 256. In particular,in preferred embodiments, such modifications are not made when the IgGconstant domain, hinge-Fc domain, hinge-Fc domain or other FcRn-bindingfragment thereof is derived from a mouse.

The amino acid modifications may be any modification, preferably at oneor more of residues 251-256, 285-290, 308-314, 385-389, and 428-436,that increases the in vivo half-life of the IgG constant domain, orFcRn-binding fragment thereof (e.g., Fc or hinge-Fc domain), and anymolecule attached thereto, and increases the affinity of the IgG, orfragment thereof, for FcRn. Preferably, the one or more modificationsalso result in a higher binding affinity of the constant domain, orFcRn-binding fragment thereof, for FcRn at pH 6.0 than at pH 7.4. Inother embodiments, the modifications alter (i.e., increase or decrease)bioavailability of the molecule, in particular, alters (i.e., increasesor decreases) transport (or concentration or half-life) of the moleculeto mucosal surfaces (e.g., of the lungs) or other portions of a targettissue. In a preferred embodiment, the amino acid modifications alter(preferably, increase) transport or concentration or half-life of themolecule to the lungs. In other embodiments, the amino acidmodifications alter (preferably, increase) transport (or concentrationor half-life) of the molecule to the heart, pancreas, liver, kidney,bladder, stomach, large or small intestine, respiratory tract, lymphnodes, nervous tissue (central and/or peripheral nervous tissue),muscle, epidermis, bone, cartilage, joints, blood vessels, bone marrow,prostate, ovary, uterine, tumor or cancer tissue, etc. In a preferredembodiment, the amino acid modifications do not abolish, or, morepreferably, do not alter, other immune effector or receptor bindingfunctions of the constant domain, for example, but not limited tocomplement fixation, ADCC and binding to FcγRI, FcγRII, and FcγRIII, ascan be determined by methods well-known and routine in the art. Inanother preferred embodiment, the modified FcRn binding fragment of theconstant domain does not contain sequences that mediate immune effectorfunctions or other receptor binding. Such fragments may be particularlyuseful for conjugation to a non-IgG or non-immunoglobulin molecule toincrease the in vivo half-life thereof. In yet another embodiment, theeffector functions are selectively altered (e.g., to reduce or increaseeffector functions).

In preferred embodiments, the amino acid modifications are substitutionsat one or more of residues 308, 309, 311, 312 and 314, particularly asubstitution with threonine at position 308, proline at position 309,serine at position 311, aspartic acid at position 312, and/or leucine atposition 314. Alternatively, the modification is the substitution withan isoleucine at position 308, proline at position 309, and/or aglutamic acid at position 311. In yet another embodiment, residues atone or more of positions 308, 309, 311, 312, and 314, are substitutedwith threonine, proline, leucine, alanine, and alanine, respectively.Accordingly, in certain embodiments the residue at position 308 issubstituted with threonine or isoleucine, the residue at position 309 issubstituted with proline, the residue at position 311 is substitutedwith serine, glutamic acid or leucine, the residue at position 312 issubstituted with alanine, and/or the residue at position 314 issubstituted with leucine or alanine. In a preferred embodiment, thesubstitution is a threonine at position 308, a proline at position 309,a serine at position 311, an aspartic acid at position 312, and/or aleucine at position 314.

In preferred embodiments, the amino acid modifications are substitutionsat one or more of residues 251, 252, 254, 255, and 256. In specificembodiments, residue 251 is substituted with leucine or arginine,residue 252 is substituted with tyrosine, phenylalanine, serine,tryptophan or threonine, residue 254 is substituted with threonine orserine, residue 255 is substituted with arginine, leucine, glycine, orisoleucine, and/or residue 256 is substituted with serine, arginine,glutamine, glutamic acid, aspartic acid, alanine, asparagine orthreonine. In a more specific embodiment, residue 251 is substitutedwith leucine, residue 252 is substituted with tyrosine, residue 254 issubstituted with threonine or serine, residue 255 is substituted witharginine, and/or residue 256 is substituted with glutamic acid.

In preferred embodiments, the amino acid modifications are substitutionsat one or more of residues 428, 433, 434, and 436. In specificembodiments, residue 428 is substituted with threonine, methionine,leucine, phenylalanine, or serine, residue 433 is substituted withlysine, arginine, serine, isoleucine, proline, glutamine or histidine,residue 434 is substituted with phenylalanine, tyrosine, or histidine,and/or residue 436 is substituted with histidine, asparagine, arginine,threonine, lysine, or methionine. In a more specific embodiment,residues at position 428 and/or 434 are substituted with methionine,and/or histidine respectively.

In preferred embodiments, the amino acid modifications are substitutionsat one or more of residues 385, 386, 387, and 389, more specifically,having substitutions at one or more of these positions. In specificembodiments, residue 385 is substituted with arginine, aspartic acid,serine, threonine, histidine, lysine, alanine or glycine, residue 386 issubstituted with threonine, proline, aspartic acid, serine, lysine,arginine, isoleucine, or methionine, residue 387 is substituted witharginine, proline, histidine, serine, threonine, or alanine, and/orresidue 389 is substituted with proline, serine or asparagine. In morespecific embodiments, residues at one or more positions 385, 386, 387,and 389 are substituted with arginine, threonine, arginine, and proline,respectively. In yet another specific embodiment, residues at one ormore positions 385, 386, and 389 are substituted with aspartic acid,proline, and serine, respectively.

In particular embodiments, amino acid modifications are made at one or acombination of residues 251, 252, 254, 255, 256, 308, 309, 311, 312,314, 385, 386, 387, 389, 428, 433, 434, and/or 436, particularly wherethe modifications are one or more of the amino acid substitutionsdescribed immediately above for these residues.

In a preferred embodiment, the molecule of the invention contains a Fcregion, or FcRn-binding domain thereof, having one or more of thefollowing substitutions: leucine at residue 251, tyrosine at residue252, threonine or serine at residue 254, arginine at residue 255,threonine at residue 308, proline at residue 309, serine at residue 311,aspartic acid at residue 312, leucine at residue 314, arginine atresidue 385, threonine at residue 386, arginine at residue 387, prolineat residue 389, methionine at residue 428, and/or tyrosine at residue434.

In a preferred embodiment, the FcRn binding domain has a substitution at1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16 or all 18 of residues 251,252, 254, 255, 256, 308, 309, 311, 312, 314, 385, 386, 387, 389, 428,433, 434, and/or 436.

Amino acid modifications can be made by any method known in the art andmany such methods are well known and routine for the skilled artisan.For example, but not by way of limitation, amino acid substitutions,deletions and insertions may be accomplished using any well-knownPCR-based technique. Amino acid substitutions may be made bysite-directed mutagenesis (see, for example, Zoller and Smith, Nucl.Acids Res. 10:6487-6500, 1982; Kunkel, Proc. Natl. Acad. Sci. USA82:488, 1985, which are hereby incorporated by reference in theirentireties). Mutants that result in increased affinity for FcRn andincreased in vivo half-life may readily be screened using well-known androutine assays, such as those described in Section 5.11, infra. In apreferred method, amino acid substitutions are introduced at one or moreresidues in the IgG constant domain or FcRn-binding fragment thereof andthe mutated constant domains or fragments are expressed on the surfaceof bacteriophage which are then screened for increased FcRn bindingaffinity (see, in particular, Section 5.2 and 5.11, infra).

Preferably, the amino acid residues to be modified are surface exposedresidues. Additionally, in making amino acid substitutions, preferablythe amino acid residue to be substituted is a conservative amino acidsubstitution, for example, a polar residue is substituted with a polarresidue, a hydrophilic residue with a hydrophilic residue, hydrophobicresidue with a hydrophobic residue, a positively charged residue with apositively charged residue, or a negatively charged residue with anegatively charged residue. Moreover, preferably, the amino acid residueto be modified is not highly or completely conserved across speciesand/or is critical to maintain the constant domain tertiary structure orto FcRn binding. For example, but not by way of limitation, modificationof the histidine at residue 310 is not preferred.

Specific mutants of the Fc domain that have increased affinity for FcRnwere isolated after the third-round panning (as described in Section 6)from a library of mutant human IgG1 molecules having mutations atresidues 308-314 (histidine at position 310 and tryptophan at position313 are fixed), those isolated after the fifth-round panning of thelibrary for residues 251-256 (isoleucine at position 253 is fixed),those isolated after fourth-round panning of the library for residues428-436 (histidine at position 429, glutamic acid at position 430,alanine at position 431, leucine at position 432, and histidine atposition 435 are fixed), and those isolated after sixth-round panning ofthe library for residues 385-389 (glutamic acid at position 388 isfixed) are listed in Table I. The wild type human IgG1 has a sequenceVal-Leu-His-Gln-Asp-Trp-Leu (SEQ ID NO:86) at positions 308-314,Leu-Met-Ile-Ser-Arg-Thr (SEQ ID NO:87) at positions 251-256,Met-His-Glu-Ala-Leu-His-Asn-His-Tyr (SEQ ID NO:88) at positions 428-436,and Gly-Gln-Pro-Glu-Asn (SEQ ID NO:89) at positions 385-389.

TABLE I MUTANTS ISOLATED BY PANNING LIBRARY MUTANTS* 251-256Leu Tyr Ile Thr Arg Glu (SEQ ID NO: 90) Leu

 Ile Ser Arg Thr (SEQ ID NO: 91) Leu

 Ile Ser Arg Ser (SEQ ID NO: 92) Leu

 Ile Ser Arg

(SEQ ID NO: 93) Leu

 Ile Ser Arg

(SEQ TD NO: 94) Leu

 Ile Ser Arg Thr (SEQ ID NO: 95) Leu Tyr Ile Ser Leu Gln (SEQ ID NO: 96)Leu Phe Ile Ser Arg Asp (SEQ ID NO: 97) Leu Phe Ile Ser Arg Thr (SEQ IDNO: 98) Leu Phe Ile Ser Arg Arg (SEQ ID NO: 99) Leu Phe Ile Thr Gly Ala(SEQ ID NO: 100) Leu Ser Ile Ser Arg Glu (SEQ ID NO: 101) Arg Thr IleSer Ile Ser (SEQ ID NO: 102) 308-314 Thr Pro  His Ser Asp Trp Leu (SEQID NO: 103) Ile Pro His Glu Asp Trp Leu (SEQ ID NO: 104) 385-389Arg Thr Arg Glu Pro (SEQ ID NO: 105)

 

 Pro Glu

(SEQ ID NO: 106) Ser Asp Pro Glu Pro (SEQ ID NO: 107) Thr Ser His GluAsn (SEQ ID NO: 108) Ser Lys Ser Glu Asn (SEQ ID NO: 109) His Arg SerGlu Asn (SEQ ID NO: 110) Lys Ile Arg Glu Asn (SEQ ID NO: 111) Gly IleThr Glu Ser (SEQ ID NO: 112) Ser Met Ala Glu Pro (SEQ ID NO: 113)428-436 Met His Glu Ala Leu

 

 His

(SEQ ID NO: 114) Met His Glu Ala Leu His Phe His His (SEQ ID NO: 115)Met His Glu Ala Leu Lys Phe His His (SEQ ID NO: 116) Met His Glu Ala LeuSer Tyr His Arg (SEQ ID NO: 117) Thr His Glu Ala Leu His Tyr His Thr(SEQ ID NO: 118) Met His Glu Ala Leu His Tyr His Tyr (SEQ ID NO: 119)*Substituting residues are indicated in bold faceThe underlined sequences in Table I correspond to sequences thatoccurred 10 to 20 times in the final round of panning and the sequencesin italics correspond to sequences that occurred 2 to 5 times in thefinal round of panning. Those sequences that are neither underlined noritalicized occurred once in the final round of panning.

In one preferred embodiment, the invention provides modifiedimmunoglobulin molecules (e.g., various antibodies) that have increasedin vivo half-life and affinity for FcRn relative to unmodified molecules(and, in preferred embodiments, altered bioavailabilty such as increasedor decreased transport to mucosal surfaces or other target tissues).Such immunoglobulin molecules include IgG molecules that naturallycontain an FcRn binding domain and other non-IgG immunoglobulins (e.g.,IgE, IgM, IgD, IgA and IgY) or fragments of immunoglobulins that havebeen engineered to contain an FcRn-binding fragment (i.e., fusionproteins comprising non-IgG immunoglobulin or a portion thereof and anFcRn binding domain). In both cases the FcRn-binding domain has one ormore amino acid modifications that increase the affinity of the constantdomain fragment for FcRn.

The modified immunoglobulins include any immunoglobulin molecule thatbinds (preferably, immunospecifically, i.e., competes off non-specificbinding), as determined by immunoassays well known in the art forassaying specific antigen-antibody binding) an antigen and contains anFcRn-binding fragment. Such antibodies include, but are not limited to,polyclonal, monoclonal, bi-specific, multi-specific, human, humanized,chimeric antibodies, single chain antibodies, Fab fragments, F(ab')₂fragments, disulfide-linked Fvs, and fragments containing either a VL orVH domain or even a complementary determining region (CDR) thatspecifically binds an antigen, in certain cases, engineered to containor fused to an FcRn binding domain.

The IgG molecules of the invention, and FcRn-binding fragments thereof,are preferably IgG1 subclass of IgGs, but may also be any other IgGsubclasses of given animals. For example, in humans, the IgG classincludes IgG1, IgG2, IgG3, and IgG4; and mouse IgG includes IgG1, IgG2a,IgG2b, IgG2c and IgG3. It is known that certain IgG subclasses, forexample, mouse IgG2b and IgG2c, have higher clearance rates than, forexample, IgG1 (Medesan et al., Eur. J. Immunol., 28:2092-2100, 1998).Thus, when using IgG subclasses other than IgG1, it may be advantageousto substitute one or more of the residues, particularly in the CH2 andCH3 domains, that differ from the IgG1 sequence with those of IgG1,thereby increasing the in vivo half-life of the other types of IgG.

The immunoglobulins (and other proteins used herein) may be from anyanimal origin including birds and mammals. Preferably, the antibodiesare human, rodent (e.g., mouse and rat), donkey, sheep, rabbit, goat,guinea pig, camel, horse, or chicken. As used herein, “human” antibodiesinclude antibodies having the amino acid sequence of a humanimmunoglobulin and include antibodies isolated from human immunoglobulinlibraries or from animals transgenic for one or more humanimmunoglobulin and that do not express endogenous immunoglobulins, asdescribed infra and, for example, in U.S. Pat. No. 5,939,598 byKucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide or may be specificfor heterologous epitopes, such as a heterologous polypeptide or solidsupport material. See, e.g., PCT publications WO 93/17715; WO 92/08802;WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol., 147:60-69, 1991;U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819;Kostelny et al., J. Immunol., 148:1547-1553, 1992.

The antibodies of the invention include derivatives that are otherwisemodified, i.e., by the covalent attachment of any type of molecule tothe antibody such that covalent attachment does not prevent the antibodyfrom binding antigen and/or generating an anti-idiotypic response. Forexample, but not by way of limitation, the antibody derivatives includeantibodies that have been modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. Any of numerous chemical modifications maybe carried out by known techniques, including, but not limited to,specific chemical cleavage, acetylation, formylation, metabolicsynthesis of tunicamycin, etc. Additionally, the derivative may containone or more non-classical amino acids.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas, pp. 563-681 (Elsevier, N.Y., 1981) (both of which areincorporated herein by reference in their entireties). The term“monoclonal antibody” as used herein is not limited to antibodiesproduced through hybridoma technology. The term “monoclonal antibody”refers to an antibody that is derived from a single clone, including anyeukaryotic, prokaryotic, or phage clone, and not the method by which itis produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In anon-limiting example, mice can be immunized with an antigen of interestor a cell expressing such an antigen. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well known techniques to any suitablemyeloma cells. Hybridomas are selected and cloned by limiting dilution.The hybridoma clones are then assayed by methods known in the art forcells that secrete antibodies capable of binding the antigen. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by inoculating mice intraperitoneally with positive hybridomaclones.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments may be producedby proteolytic cleavage of immunoglobulin molecules, using enzymes suchas papain (to produce Fab fragments) or pepsin (to produce F(ab′)₂fragments). F(ab′)₂ fragments contain the complete light chain, and thevariable region, the CH1 region and the hinge region of the heavy chain.

For example, antibodies can also be generated using various phagedisplay methods known in the art. In phage display methods, functionalantibody domains are displayed on the surface of phage particles whichcarry the polynucleotide sequences encoding them. In a particularembodiment, such phage can be utilized to display antigen bindingdomains, such as Fab and Fv or disulfide-bond stabilized Fv, expressedfrom a repertoire or combinatorial antibody library (e.g., human ormurine). Phage expressing an antigen binding domain that binds theantigen of interest can be selected or identified with antigen, e.g.,using labeled antigen or antigen bound or captured to a solid surface orbead. Phage used in these methods are typically filamentous phage,including fd and M13. The antigen binding domains are expressed as arecombinantly fused protein to either the phage gene III or gene VIIIprotein. Alternatively, the modified FcRn binding portion ofimmunoglobulins of the present invention can be also expressed in aphage display system. Examples of phage display methods that can be usedto make the immunoglobulins, or fragments thereof, of the presentinvention include those disclosed in Brinkman et al., J. Immunol.Methods, 182:41-50, 1995; Ames et al., J. Immunol. Methods, 184:177-186,1995; Kettleborough et al., Eur. J. Immunol., 24:952-958, 1994; Persicet al., Gene, 187:9-18, 1997; Burton et al., Advances in Immunology,57:191-280, 1994; PCT application No. PCT/GB91/01134; PCT publicationsWO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired fragments, and expressed in any desired host, includingmammalian cells, insect cells, plant cells, yeast, and bacteria, e.g.,as described in detail below. For example, techniques to recombinantlyproduce Fab, Fab' and F(ab')₂ fragments can also be employed usingmethods known in the art such as those disclosed in PCT publication WO92/22324; Mullinax et al., BioTechniques, 12(6):864-869, 1992; and Sawaiet al., AJRI, 34:26-34, 1995; and Better et al., Science, 240:1041-1043,1988 (each of which is incorporated by reference in its entirety).Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology, 203:46-88, 1991; Shu etal., PNAS, 90:7995-7999, 1993; and Skerra et al., Science,240:1038-1040, 1988.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use chimeric, humanized,or human antibodies. A chimeric antibody is a molecule in whichdifferent portions of the antibody are derived from different animalspecies, such as antibodies having a variable region derived from amurine monoclonal antibody and a constant region derived from a humanimmunoglobulin. Methods for producing chimeric antibodies are known inthe art. See e.g., Morrison, Science, 229:1202, 1985; Oi et al.,BioTechniques, 4:214 1986; Gillies et al., J. Immunol. Methods,125:191-202, 1989; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397,which are incorporated herein by reference in their entireties.Humanized antibodies are antibody molecules from non-human species thatbind the desired antigen having one or more complementarity determiningregions (CDRs) from the non-human species and framework regions from ahuman immunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature, 332:323, 1988, which areincorporated herein by reference in their entireties. Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology, 28(4/5):489-498,1991; Studnicka et al., Protein Engineering, 7(6):805-814, 1994; Roguskaet al., Proc Natl. Acad. Sci. USA, 91:969-973, 1994), and chainshuffling (U.S. Pat. No. 5,565,332), all of which are herebyincorporated by reference in their entireties.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO96/34096; WO 96/33735; and WO 91/10741, each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For an overview of thistechnology for producing human antibodies, see Lonberg and Huszar, Int.Rev. Immunol., 13:65-93, 1995. For a detailed discussion of thistechnology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g., PCTpublications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735;European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793;5,916,771; and 5,939,598, which are incorporated by reference herein intheir entireties. In addition, companies such as Abgenix, Inc.(Freemont, Calif.), Medarex (NJ) and Genpharm (San Jose, Calif.) can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology,12:899-903, 1988).

In particular embodiments, the modified antibodies have in vivotherapeutic and/or prophylactic uses. Examples of therapeutic andprophylactic antibodies which may be so modified include, but are notlimited to, SYNAGIS® (MedImmune, MD) which is a humanizedanti-respiratory syncytial virus (RSV) monoclonal antibody for thetreatment of patients with RSV infection; HERCEPTIN® (Trastuzumab)(Genentech, CA) which is a humanized anti-HER2 monoclonal antibody forthe treatment of patients with metastatic breast cancer; REMICADE®(infliximab) (Centocor, Pa.) which is a chimeric anti-TNFα monoclonalantibody for the treatment of patients with Crone's disease; REOPRO®(abciximab) (Centocor) which is an anti-glycoprotein IIb/IIIa receptoron the platelets for the prevention of clot formation; ZENAPAX®(daclizumab) (Roche Pharmaceuticals, Switzerland) which is animmunosuppressive, humanized anti-CD25 monoclonal antibody for theprevention of acute renal allograft rejection. Other examples are ahumanized anti-CD18 F(ab')₂ (Genentech); CDP860 which is a humanizedanti-CD18 F(ab')₂ (Celltech, UK); PRO542 which is an anti-HIV gp120antibody fused with CD4 (Progenics/Genzyme Transgenics); Ostavir whichis a human anti Hepatitis B virus antibody (Protein DesignLab/Novartis); PROTOVIR™ which is a humanized anti-CMV IgG1 antibody(Protein Design Lab/Novartis); MAK-195 (SEGARD) which is a murineanti-TNF-α F(ab′)₂ (Knoll Pharma/BASF); IC14 which is an anti-CD14antibody (ICOS Pharm); a humanized anti-VEGF IgG1 antibody (Genentech);OVAREX™ which is a murine anti-CA 125 antibody (Altarex); PANOREX™ whichis a murine anti-17-IA cell surface antigen IgG2a antibody (GlaxoWellcome/Centocor); BEC2 which is a murine anti-idiotype (GD3 epitope)IgG antibody (ImClone System); IMC-C225 which is a chimeric anti-EGFRIgG antibody (ImClone System); VITAXIN™ which is a humanized anti-αVβ3integrin antibody (Applied Molecular Evolution/MedImmune); Campath1H/LDP-03 which is a humanized anti CD52 IgG1 antibody (Leukosite);Smart M195 which is a humanized anti-CD33 IgG antibody (Protein DesignLab/Kanebo); RITUXAN™ which is a chimeric anti-CD20 IgG1 antibody (IDECPharm/Genentech, Roche/Zettyaku); LYMPHOCIDE™ which is a humanizedanti-CD22 IgG antibody (Immunomedics); Smart ID10 which is a humanizedanti-HLA antibody (Protein Design Lab); ONCOLYM™ (Lym-1) is aradiolabelled murine anti-HLA DIAGNOSTIC REAGENT antibody (Techniclone);ABX-IL8 is a human anti-IL8 antibody (Abgenix); anti-CD11a is ahumanized IgG1 antibody (Genetech/Xoma); ICM3 is a humanized anti-ICAM3antibody (ICOS Pharm); IDEC-114 is a primatied anti-CD80 antibody (IDECPharm/Mitsubishi); ZEVALIN™ is a radiolabelled murine anti-CD20 antibody(IDEC/Schering AG); IDEC-131 is a humanized anti-CD40L antibody(IDEC/Eisai); IDEC-151 is a primatized anti-CD4 antibody (IDEC);IDEC-152 is a primatized anti-CD23 antibody (IDEC/Seikagaku); SMARTanti-CD3 is a humanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is ahumanized anti-complement factor 5 (C5) antibody (Alexion Pharm); D2E7is a humanized anti-TNF-α antibody (CAT/BASF); CDP870 is a humanizedanti-TNF-α Fab fragment (Celltech); IDEC-151 is a primatized anti-CD4IgG1 antibody (IDEC Pharm/SmithKline Beecham); MDX-CD4 is a humananti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571 is a humanizedanti-TNF-α IgG4 antibody (Celltech); LDP-02 is a humanized anti-α4137antibody (LeukoSite/Genentech); OrthoClone OKT4A is a humanized anti-CD4IgG antibody (Ortho Biotech); ANTOVAT™ is a humanized anti-CD40L IgGantibody (Biogen); ANTEGREN™ is a humanized anti-VLA-4 IgG antibody(Elan); MDX-33 is a human anti-CD64 (FcγR) antibody (Medarex/Centeon);SCH55700 is a humanized anti-IL-5 IgG4 antibody (Celltech/Schering);SB-240563 and SB-240683 are humanized anti-IL-5 and IL-4 antibodies,respectively, (SmithKline Beecham); rhuMab-E25 is a humanized anti-IgEIgG1 antibody (Genentech/Norvartis/Tanox Biosystems); IDEC-152 is aprimatized anti-CD23 antibody (IDEC Pharm); ABX-CBL is a murine antiCD-147 IgM antibody (Abgenix); BTI-322 is a rat anti-CD2 IgG antibody(Medimmune/Bio Transplant); Orthoclone/OKT3 is a murine anti-CD3 IgG2aantibody (ortho Biotech); SIMULECTT™ is a chimeric anti-CD25 IgG1antibody (Novartis Pharm); LDP-01 is a humanized anti-β₂-integrin IgGantibody (LeukoSite); Anti-LFA-1 is a murine anti CD18 F(ab′)₂(Pasteur-Merieux/Immunotech); CAT-152 is a human anti-TGF-β₂ antibody(Cambridge Ab Tech); and Corsevin M is a chimeric anti-Factor VIIantibody (Centocor).

In specific embodiments, the invention provides modified antibodieshaving one or more of the mutations described herein and thatimmunospecifically bind RSV, e.g., SYNAGIS®. The present invention alsoprovides modified antibodies having one or more of the mutationsdescribed herein and that comprise a variable heavy (VH) and/or variablelight (VL) domain having the amino acid sequence of any VH and/or VLdomain listed in Table III. The present invention further encompassesanti-RSV antibodies comprising one or more VH complementaritydetermining regions (CDRs) and/or one or more VL CDRs having the aminoacid sequence of one or more VH CDRs and/or VL CDRS listed in Table IIIor one or more of the CDRs listed in Table II wherein one or more of thebolded and underlined residues has an amino acid substitution,preferably that increases the affinity of the antibody for RSV. Inspecific embodiments, the antibody to be modified is AFFF, p12f2, p12f4,p11d4, Ale109, A12a6, A13c4, A17d4, A4B4, A8C7, 1X-493L1FR, H3-3F4,M3H9, Y10H6, DG, AFFF(1), 6H8, L1-7E5, L215B10, A13A11, A1H5, A4B4(1),A4B4L1FR-S28R, A4B4-F52S.

TABLE II CDR Sequences of SYNAGIS ® CDR Sequence SEQ ID NO: VH1 T SGMSVG 1 VH2 DIWWD D K KD YNPSLK S 2 VH3 S MI T N W YFDV 3 VL1 KCQLSVGYMH 4 VL2 DT SKLA S 5 VL3 FQGS G YP F T 6

ANTI-RSV ANTIBODIES Antibody Name VH Domain VH CDR1 VH CDR2 VH CDR3 VLDomain VL CDR1 VL CDR2 VL CDR3 SYNAGIS SEQ ID NO: 7 TSGMSVGDIWWDDKKDYNPSLKS SMITNWYFDV SEQ ID NO: 8 KCQLSVGYMH DTSKLAS FQGSGYPFT(SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 4) (SEQ ID NO:5) (SEQ ID NO: 6) AFFF SEQ ID NO: 9 T A GMSVG DIWWDDKKDYNPSLKS SMITN FYFDV SEQ ID NO: 13 SASS SVGYMH DT F KLAS FQ F SGYPFT (SEQ ID NO: 10)(SEQ ID NO: 2) (SEQ ID NO: 12) (SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ IDNO: 16) p12f2 SEQ ID NO: 17 T P GMSVG DIWWDDKK H YNPSLK D D MI F N FYFDV SEQ ID NO: 21 SLSSR VGYMH DT FY L S S FQGSGYPFT (SEQ ID NO: 18)(SEQ ID NO: 19) (SEQ ID NO: 20) (SEQ ID NO: 22) (SEQ ID NO: 23) (SEQ IDNO: 6) p12f4 SEQ ID NO: 24 T P GMSVG DIWWD G KK H YNPSLK D D MI F N FYFDV SEQ ID NO: 26 SLSSR VGYMH DT RG L P S FQGSGYPFT (SEQ ID NO: 18)(SEQ ID NO: 25) (SEQ ID NO: 20) (SEQ ID NO: 22) (SEQ ID NO: 27) (SEQ IDNO: 6) p11d4 SEQ ID NO: 28 T P GMSVG DIWWD G KK H YNPSLK D D MI F NWYFDVSEQ ID NO: 30 SPSSR VGYMH DT MR LAS FQGSGYPFT (SEQ ID NO: 18) (SEQ IDNO: 25) (SEQ ID NO: 29) (SEQ ID NO: 31) (SEQ ID NO: 32) (SEQ ID NO: 6)Ale109 SEQ ID NO: 33 T A GMSVG DIWWD G KK H YNPSLK D D MI F NWYFDV SEQID NO: 34 SLSSR VGYMH DT F KL S S FQGSGYPFT (SEQ ID NO: 10) (SEQ ID NO:25) (SEQ ID NO: 29) (SEQ ID NO: 22) (SEQ ID NO: 35) (SEQ ID NO: 6) A12a6SEQ ID NO: 36 T A GMSVG DIWWD G KKDYNPSLK D D MI F N F YFDV SEQ ID NO:38 SASSR VGYMH DT F KL S S FQGSGYPFT (SEQ ID NO: 10) (SEQ ID NO: 37)(SEQ ID NO: 20) (SEQ ID NO: 39) (SEQ ID NO: 35) (SEQ ID NO: 6) A13c4 SEQID NO: 40 T A GMSVG DIWWD G KK S YNPSLK D D MI F N F YFDV SEQ ID NO: 42SLSSR VGYMH DT MYQS S FQGSGYPFT (SEQ ID NO: 10) (SEQ ID NO: 41) (SEQ IDNO: 20) (SEQ ID NO: 22) (SEQ ID NO: 43) (SEQ ID NO: 6) A17d4 SEQ ID NO:44 T A GMSVG DIWWDDKK S YNPSLK D D MI F N F YFDV SEQ ID NO: 46 LPSSRVGYMH DT MYQS S FQGSGYPFT (SEQ ID NO: 10) (SEQ ID NO: 45) (SEQ ID NO: 20(SEQ ID NO: 47) (SEQ ID NO: 43) (SEQ ID NO: 6) A4B4 SEQ ID NO: 48 T AGMSVG DIWWDDKK H YNPSLK D D MI F N F YFDV SEQ ID NO: 49 SASSR VGYMH DTFF L D S FQGSGYPFT (SEQ ID NO: 10) (SEQ ID NO: 19) (SEQ ID NO: 20) (SEQID NO: 39) (SEQ ID NO: 50) (SEQ ID NO: 6) A8C7 SEQ ID NO: 51 T A GMSVGDIWWDDKK S YNPSLK D D MI F NWYFDV SEQ ID NO: 52 SPSSR VGYMH DT RYQS SFQGSGYPFT (SEQ ID NO: 10) (SEQ ID NO: 45) (SEQ ID NO: 29) (SEQ ID NO:31) (SEQ ID NO: 53) (SEQ ID NO: 6) 1X-493L1FR SEQ ID NO: 7 TSGMSVGDIWWDDKKDYNPSLKS SMITNWYFDV SEQ ID NO: 54 SASS SVGYMH DTSKLAS FQGSGYPFT(SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 14) (SEQ ID NO:5) (SEQ ID NO: 6) H3-3F4 SEQ ID NO: 55 T A GMSVG DIWWDDKKDYNPSLKS D MI FNWYFDV SEQ ID NO: 56 SASS VGYMH DT F KLAS FQGSGYPFT (SEQ ID NO: 10) (SEQID NO: 2) (SEQ ID NO: 29) (SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 6)M3H9 SEQ ID NO: 55 T A GMSVG DIWWDDKKDYNPSLKS D MI F NWYFDV SEQ ID NO:124 SASS SVGYMH DT Y K QT S FQGSGYPFT (SEQ ID NO: 10) (SEQ ID NO: 2)(SEQ ID NO: 29) (SEQ ID NO: 14) (SEQ ID NO: 57) (SEQ ID NO: 6) Y10H6 SEQID NO: 55 T A GMSVG DIWWDDKKDYNPSLKS D MI F NWYFDV SEQ ID NO: 58 SASSSVGYMH DT RY L S S FQGSGYPFT (SEQ ID NO: 10) (SEQ ID NO: 2) (SEQ ID NO:29) (SEQ ID NO: 14) (SEQ ID NO: 59) (SEQ ID NO: 6) DG SEQ ID NO: 78 T AGMSVG DIWWDDKKDYNPSLKS D MITN F YFDV SEQ ID NO: 56 SASS SVGYMH DT F KLASFQGSGYPFT (SEQ ID NO: 10) (SEQ ID NO: 2) (SEQ ID NO: 79) (SEQ ID NO: 14)(SEQ ID NO: 15) (SEQ ID NO: 6) AFFF(1) SEQ ID NO: 9 T A GMSVGDIWWDDKKDYNPSLKS S MITN F YFDV SEQ ID NO: 60 SASS SVGYMH DT F KLAS FQGSF YPFT (SEQ ID NO: 10) (SEQ ID NO: 2) (SEQ ID NO: 12) (SEQ ID NO: 14)(SEQ ID NO: 15) (SEQ ID NO: 61) 6H8 SEQ ID NO: 78 T A GMSVGDIWWDDKKDYNPSLKS D MITN F YFDV SEQ ID NO: 62 SASS SVGYMH DT F KL T SFQGSGYPFT (SEQ ID NO: 10) (SEQ ID NO: 2) (SEQ ID NO: 79) (SEQ ID NO: 14)(SEQ ID NO: 63) (SEQ ID NO: 6) L1-7E5 SEQ ID NO: 78 T A GMSVGDIWWDDKKDYNPSLKS D MITN F YFDV SEQ ID NO: 64 SASSR VGYMH DT F KLASFQGSGYPFT (SEQ ID NO: 10) (SEQ ID NO: 2) (SEQ ID NO: 79) (SEQ ID NO: 39)(SEQ ID NO: 15) (SEQ ID NO: 6) L215B10 SEQ ID NO: 78 T A GMSVGDIWWDDKKDYNPSLKS D MITN F YFDV SEQ ID NO: 65 SASS SVGYMH DT FR LASFQGSGYPFT (SEQ ID NO: 10) (SEQ ID NO: 2) (SEQ ID NO: 79) (SEQ ID NO: 14)(SEQ ID NO: 66) (SEQ ID NO: 6) A13A11 SEQ ID NO: 67 T A GMSVG DIWWDDKK HYNPSLK D D MI F NWYFDV SEQ ID NO: 68 SPSSR VGYMH DT YRHS S FQGSGYPFT(SEQ ID NO: 10) (SEQ ID NO: 19) (SEQ ID NO: 29) (SEQ ID NO: 31) (SEQ IDNO: 69) (SEQ ID NO: 6) A1H5 SEQ ID NO: 70 T A GMSVG DIWWD G KK H YNPSLKD D MI F NWYFDV SEQ ID NO: 71 SLSSS VGYMH DT FFHR S FQGSGYPFT (SEQ IDNO: 10) (SEQ ID NO: 25) (SEQ ID NO: 29) (SEQ ID NO: 72) (SEQ ID NO: 73)(SEQ ID NO: 6) A4B4(1) SEQ ID NO: 48 T A GMSVG DIWWDDKK H YNPSLK D D MIF N F YFDV SEQ ID NO: 74 SASSR VGYMH DT LL L D S FQGSGYPFT (SEQ ID NO:10) (SEQ ID NO: 19) (SEQ ID NO: 20) (SEQ ID NO: 39) (SEQ ID NO: 75) (SEQID NO: 6) A4B4L1F SEQ ID NO: 48 T A GMSVG DIWWDDKK H YNPSLK D D MI F N FYFDV SEQ ID NO: 11 SASSR VGYMH DTSKLAS FQGSGYPFT R-S28R (SEQ ID NO: 10)(SEQ ID NO: 19) (SEQ ID NO: 20) (SEQ ID NO: 39) (SEQ ID NO: 5) (SEQ IDNO: 6) A4B4- SEQ ID NO: 48 T A GMSVG DIWWDDKK H YNPSLK D D MI F N F YFDVSEQ ID NO: 76 SASSR VGYMH DTS F L D S FQGSGYPFT F52S (SEQ ID NO: 10)(SEQ ID NO: 19) (SEQ ID NO: 20) (SEQ ID NO: 39) (SEQ ID NO: 77) (SEQ IDNO: 6)

In other embodiments, the antibody is a modified anti-α_(v)β₃ antibody,preferably a Vitaxin antibody (see, PCT publications WO 98/33919 and WO00/78815, both by Huse et al., and both of which are incorporated byreference herein in their entireties).

Modified IgGs of the present invention having longer half-lives thanwild type may also include IgGs whose bioactive sites, such asantigen-binding sites, Fc-receptor binding sites, or complement-bindingsites, are modified by genetic engineering to increase or reduce suchactivities compared to the wild type.

Modification of these and other therapeutic antibodies to increase thein vivo half-life permits administration of lower effective dosagesand/or less frequent dosing of the therapeutic antibody. Suchmodification to increase in vivo half-life can also be useful to improvediagnostic immunoglobulins as well, for example, permittingadministration of lower doses to achieve sufficient diagnosticsensitivity.

The present invention also provides fusion proteins comprising abioactive molecule and an hinge-Fc region or a fragment thereof(preferably human) having one or more modifications (i.e.,substitutions, deletions, or insertions) in amino acid residuesidentified to be involved in the interaction between the hinge-Fc regionand the FcRn receptor. In particular, the present invention providesfusion proteins comprising a bioactive molecule recombinantly fused orchemically conjugated (including both covalent and non-covalentconjugations) to a CH2 domain having one or more modifications in aminoacid residues 251-256, 285-290, and/or amino acid residues 308-314,and/or to a CH3 domain having one or more modifications in amino acidresidues 385-389 and/or 428-436, in particular, one or more of the aminoacid substitutions discussed above. The fusion of a bioactive moleculeto a constant domain or a fragment thereof with one or more of suchmodifications increases the in vivo half-life of the bioactive molecule.

In a preferred embodiment, fusion proteins of the invention comprise abioactive molecule recombinantly fused or chemically conjugated to a CH2domain having one or more amino acid residue substitutions in amino acidresidues 251-256, 285-290, and/or amino acid residues 308-314, and/or toa CH3 domain having one or more modifications in amino acid residues385-389 and/or 428-436. In certain embodiments, a fusion proteincomprises a CH2 domain of IgG molecule in which amino acid residues 253,310, and 313 are not modified. In another embodiments, a fusion proteincomprises a CH3 domain of IgG molecule in which amino acid residues 388,429, 430, 431, 432, and 435 are not modified.

A bioactive molecule can be any polypeptide or synthetic drug known toone of skill in the art. Preferably, a bioactive molecule is apolypeptide consisting of at least 5, preferably at least 10, at least20, at least 30, at least 40, at least 50, at least 60, at least 70, atleast 80, at least 90 or at least 100 amino acid residues. Examples ofbioactive polypeptides include, but are not limited to, various types ofantibodies, cytokines (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10,IL-12, IL-15, IFN-γ, IFN-α, and IFN-β), cell adhesion molecules (e.g.,CTLA4, CD2, and CD28), ligands (e.g., TNF-α, TNF-β, and ananti-angiogenic factor such as endostatin), receptors, antibodies andgrowth factors (e.g., PDGF, EGF, NGF, and KGF).

A bioactive molecule can also be a therapeutic moiety such as acytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agentor a radioactive element (e.g., alpha-emitters, gamma-emitters, etc.).Examples of cytostatic or cytocidal agents include, but are not limitedto, paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The present invention also provides polynucleotides comprising anucleotide sequence encoding a modified IgG of the invention andfragments thereof which contain the modified FcRn binding sites withincreased affinity and vectors comprising said polynucleotides.Furthermore, the invention includes polynucleotides that hybridize understringent or lower stringent hybridization conditions to polynucleotidesencoding modified IgGs of the present invention.

The nucleotide sequence of modified IgGs and the polynucleotidesencoding the same may be obtained by any methods known in the art,including general DNA sequencing method, such as dideoxy chaintermination method (Sanger sequencing), and oligonucleotide priming incombination with PCR, respectively.

5.2 Identification of Mutations Within the Hinge-Fc Region ofImmunoglobulin Molecules

One or more modifications in amino acid residues 251-256, 285-290,308-314, 385-389, and 428-436 of the constant domain may be introducedutilizing any technique known to those of skill in the art. The constantdomain or fragment thereof having one or more modifications in aminoacid residues 251-256, 285-290, 308-314, 385-389, and 428-436 may bescreened by, for example, a binding assay to identify the constantdomain or fragment thereof with increased affinity for the FcRn receptor(e.g., as described in section 5.11, infra). Those modifications in thehinge-Fc domain or the fragments thereof which increase the affinity ofthe constant domain or fragment thereof for the FcRn receptor can beintroduced into antibodies to increase the in vivo half-lives of saidantibodies. Further, those modifications in the constant domain or thefragment thereof which increase the affinity of the constant domain orfragment thereof for the FcRn can be fused to bioactive molecules toincrease the in vivo half-lives of said bioactive molecules (and,preferably alter (increase or decrease) the bioavailability of themolecule, for example, to increase or decrease transport to mucosalsurfaces (or other target tissue) (e.g., the lungs).

5.2.1 Mutagenesis

Mutagenesis may be performed in accordance with any of the techniquesknown in the art including, but not limited to, synthesizing anoligonucleotide having one or more modifications within the sequence ofthe constant domain of an antibody or a fragment thereof (e.g., the CH2or CH3 domain) to be modified. Site-specific mutagenesis allows theproduction of mutants through the use of specific oligonucleotidesequences which encode the DNA sequence of the desired mutation, as wellas a sufficient number of adjacent nucleotides, to provide a primersequence of sufficient size and sequence complexity to form a stableduplex on both sides of the deletion junction being traversed.Typically, a primer of about 17 to about 75 nucleotides or more inlength is preferred, with about 10 to about 25 or more residues on bothsides of the junction of the sequence being altered. A number of suchprimers introducing a variety of different mutations at one or morepositions may be used to generated a library of mutants.

The technique of site-specific mutagenesis is well known in the art, asexemplified by various publications (see, e.g., Kunkel et al., MethodsEnzymol., 154:367-82, 1987, which is hereby incorporated by reference inits entirety). In general, site-directed mutagenesis is performed byfirst obtaining a single-stranded vector or melting apart of two strandsof a double stranded vector which includes within its sequence a DNAsequence which encodes the desired peptide. An oligonucleotide primerbearing the desired mutated sequence is prepared, generallysynthetically. This primer is then annealed with the single-strandedvector, and subjected to DNA polymerizing enzymes such as T7 DNApolymerase, in order to complete the synthesis of the mutation-bearingstrand. Thus, a heteroduplex is formed wherein one strand encodes theoriginal non-mutated sequence and the second strand bears the desiredmutation. This heteroduplex vector is then used to transform ortransfect appropriate cells, such as E. coli cells, and clones areselected which include recombinant vectors bearing the mutated sequencearrangement. As will be appreciated, the technique typically employs aphage vector which exists in both a single stranded and double strandedform. Typical vectors useful in site-directed mutagenesis includevectors such as the M13 phage. These phage are readily commerciallyavailable and their use is generally well known to those skilled in theart. Double stranded plasmids are also routinely employed in sitedirected mutagenesis which eliminates the step of transferring the geneof interest from a plasmid to a phage.

Alternatively, the use of PCR™ with commercially available thermostableenzymes such as Tag DNA polymerase may be used to incorporate amutagenic oligonucleotide primer into an amplified DNA fragment that canthen be cloned into an appropriate cloning or expression vector. See,e.g., Tomic et al., Nucleic Acids Res., 18(6):1656, 1987, and Upender etal., Biotechniques, 18(1):29-30, 32, 1995, for PCR™-mediated mutagenesisprocedures, which are hereby incorporated in their entireties. PCR™employing a thermostable ligase in addition to a thermostable polymerasemay also be used to incorporate a phosphorylated mutagenicoligonucleotide into an amplified DNA fragment that may then be clonedinto an appropriate cloning or expression vector (see e.g., Michael,Biotechniques, 16(3):410-2, 1994, which is hereby incorporated byreference in its entirety).

Other methods known to those of skill in art of producing sequencevariants of the Fc domain of an antibody or a fragment thereof can beused. For example, recombinant vectors encoding the amino acid sequenceof the constant domain of an antibody or a fragment thereof may betreated with mutagenic agents, such as hydroxylamine, to obtain sequencevariants.

5.2.2 Panning

Vectors, in particular, phage, expressing constant domains or fragmentsthereof having one or more modifications in amino acid residues 251-256,285-290, 308-314, 385-389, and/or 428-436 can be screened to identifyconstant domains or fragments thereof having increased affinity for FcRnto select out the highest affinity binders from a population of phage.Immunoassays which can be used to analyze binding of the constant domainor fragment thereof having one or more modifications in amino acidresidues 251-256, 285-290, 308-314, 385-389, and/or 428-436 to the FcRninclude, but are not limited to, radioimmunoassays, ELISA (enzyme linkedimmunosorbent assay), “sandwich” immunoassays, and fluorescentimmunoassays. Such assays are routine and well known in the art (see,e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated byreference herein in its entirety). Exemplary immunoassays are describedbriefly herein below (but are not intended by way of limitation).BIAcore kinetic analysis can also be used to determine the binding onand off rates of a constant domain or a fragment thereof having one ormore modifications in amino acid residues 251-256, 285-290, 308-314,385-389, and/or 428-436 to the FcRn. BIAcore kinetic analysis comprisesanalyzing the binding and dissociation of a constant domain or afragment thereof having one or more modifications in amino acid residues251-256, 285-290, 308-314, 385-389, and/or 428-436 from chips withimmobilized FcRn on their surface (see section 5.1 and the Examplesection infra).

5.2.3 Sequencing

Any of a variety of sequencing reactions known in the art can be used todirectly sequence the nucleotide sequence encoding constant domains orfragments thereof having one or more modifications in amino acidresidues 251-256, 285-290, 308-314, 385-389, and/or 428-436. Examples ofsequencing reactions include those based on techniques developed byMaxim and Gilbert (Proc. Natl. Acad. Sci. USA, 74:560, 1977) or Sanger(Proc. Natl. Acad. Sci. USA, 74:5463, 1977). It is also contemplatedthat any of a variety of automated sequencing procedures can be utilized(Bio/Techniques, 19:448, 1995), including sequencing by massspectrometry (see, e.g., PCT Publication No. WO 94/16101, Cohen et al.,Adv. Chromatogr., 36:127-162, 1996, and Griffin et al., Appl. Biochem.Biotechnol., 38:147-159, 1993).

5.3 Recombinant Methods of Producing Antibodies

The antibodies of the invention or fragments thereof can be produced byany method known in the art for the synthesis of antibodies, inparticular, by chemical synthesis or preferably, by recombinantexpression techniques.

The nucleotide sequence encoding an antibody may be obtained from anyinformation available to those of skill in the art (i.e., from Genbank,the literature, or by routine cloning). If a clone containing a nucleicacid encoding a particular antibody or an epitope-binding fragmentthereof is not available, but the sequence of the antibody molecule orepitope-binding fragment thereof is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A⁺RNA, isolated from any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody) by PCR amplification using synthetic primershybridizable to the 3′ and 5′ ends of the sequence or by cloning usingan oligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence of the antibody is determined, thenucleotide sequence of the antibody may be manipulated using methodswell known in the art for the manipulation of nucleotide sequences,e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.(see, for example, the techniques described in Sambrook et al., 1990,Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.; and Ausubel et al., eds., 1998,Current Protocols in Molecular Biology, John Wiley & Sons, NY, which areboth incorporated by reference herein in their entireties), to generateantibodies having a different amino acid sequence by, for example,introducing amino acid substitutions, deletions, and/or insertions intothe epitope-binding domain regions of the antibodies and preferably,into the hinge-Fc regions of the antibodies which are involved in theinteraction with the FcRn. In a preferred embodiment, antibodies havingone or more modifications in amino acid residues 251-256, 285-290,308-314, 385-389, and 428-436 are generated.

Recombinant expression of an antibody requires construction of anexpression vector containing a nucleotide sequence that encodes theantibody. Once a nucleotide sequence encoding an antibody molecule or aheavy or light chain of an antibody, or portion thereof (preferably, butnot necessarily, containing the heavy or light chain variable region)has been obtained, the vector for the production of the antibodymolecule may be produced by recombinant DNA technology using techniqueswell known in the art. Thus, methods for preparing a protein byexpressing a polynucleotide containing an antibody encoding nucleotidesequence are described herein. Methods which are well known to thoseskilled in the art can be used to construct expression vectorscontaining antibody coding sequences and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. The invention, thus, provides replicable vectorscomprising a nucleotide sequence encoding the constant region of theantibody molecule with one or more modifications in the amino acidresidues involved in the interaction with the FcRn (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464). The nucleotide sequence encoding the heavy-chain variableregion, light-chain variable region, both the heavy-chain andlight-chain variable regions, an epitope-binding fragment of the heavy-and/or light-chain variable region, or one or more complementaritydetermining regions (CDRs) of an antibody may be cloned into such avector for expression.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody having an increased affinity for theFcRn and an increased in vivo half-life. Thus, the invention includeshost cells containing a polynucleotide encoding an antibody, a constantdomain or a FcRn binding fragment thereof having one or moremodifications in amino acid residues 251-256, 285-290, 308-314, 385-389,and/or 428-436, preferably, operably linked to a heterologous promoter.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude, but are not limited to, microorganisms such as bacteria (e.g.,E. coli and B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces and Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; and tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; and mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 and NSO cells) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter). Preferably, bacterial cells such as Escherichiacoli, and more preferably, eukaryotic cells, especially for theexpression of whole recombinant antibody molecule, are used for theexpression of a recombinant antibody molecule. For example, mammaliancells such as Chinese hamster ovary cells (CHO), in conjunction with avector such as the major intermediate early gene promoter element fromhuman cytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene, 45:101, 1986, and Cockett et al.,Bio/Technology, 8:2, 1990).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited to,the E. coli expression vector pUR278 (Ruther et al., EMBO, 12:1791,1983), in which the antibody coding sequence may be ligated individuallyinto the vector in frame with the lacZ coding region so that a fusionprotein is produced; and pIN vectors (Inouye & Inouye, Nucleic AcidsRes., 13:3101-3109, 1985 and Van Heeke & Schuster, J. Biol. Chem.,24:5503-5509, 1989).

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized to express an antibody molecule of the invention. In caseswhere an adenovirus is used as an expression vector, the antibody codingsequence of interest may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingthe antibody molecule in infected hosts (e.g., see Logan & Shenk, Proc.Natl. Acad. Sci. USA, 81:355-359, 1984). Specific initiation signals mayalso be required for efficient translation of inserted antibody codingsequences. These signals include the ATG initiation codon and adjacentsequences. Furthermore, the initiation codon must be in phase with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see, e.g., Bitter et al., Methods in Enzymol.,153:516-544, 1987).

In addition, a host cell strain may be chosen which modulates theexpression of the antibody sequences, or modifies and processes theantibody in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the antibody. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the antibody expressed. To this end,eukaryotic host cells which possess the cellular machinery for properprocessing of the primary transcript, glycosylation, and phosphorylationof the gene product may be used. Such mammalian host cells include butare not limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, andin particular, myeloma cells such as NSO cells, and related cell lines,see, for example, Morrison et al., U.S. Pat. No. 5,807,715, which ishereby incorporated by reference in its entirety.

For long-term, high-yield production of recombinant antibodies, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compositions that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited to,the herpes simplex virus thymidine kinase (Wigler et al., Cell, 11:223,1977), hypoxanthineguanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA, 48:202, 1992), and adeninephosphoribosyltransferase (Lowy et al., Cell, 22:8-17, 1980) genes canbe employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA, 77:357, 1980 and O'Hare et al., Proc.Natl. Acad. Sci. USA, 78:1527, 1981); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072,1981); neo, which confers resistance to the aminoglycoside G-418 (Wu andWu, Biotherapy, 3:87-95, 1991; Tolstoshev, Ann. Rev. Pharmacol.Toxicol., 32:573-596, 1993; Mulligan, Science, 260:926-932, 1993; andMorgan and Anderson, Ann. Rev. Biochem., 62: 191-217, 1993; and May, TIBTECH, 11(5):155-215, 1993); and hygro, which confers resistance tohygromycin (Santerre et al., Gene, 30:147, 1984). Methods commonly knownin the art of recombinant DNA technology may be routinely applied toselect the desired recombinant clone, and such methods are described,for example, in Ausubel et al. (eds.), 1993, Current Protocols inMolecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transferand Expression, A Laboratory Manual, Stockton Press, NY; in Chapters 12and 13, Dracopoli et al. (eds), 1994, Current Protocols in HumanGenetics, John Wiley & Sons, NY; and Colberre-Garapin et al., J. Mol.Biol., 150:1, 1981, which are incorporated by reference herein in theirentireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, 1987, The useof vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3. Academic Press, NewYork). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol., Cell. Biol.,3:257, 1983).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides or different selectablemarkers to ensure maintenance of both plasmids. Alternatively, a singlevector may be used which encodes, and is capable of expressing, bothheavy and light chain polypeptides. In such situations, the light chainshould be placed before the heavy chain to avoid an excess of toxic freeheavy chain (Proudfoot, Nature, 322:52, 1986; and Kohler, Proc. Natl.Acad. Sci. USA, 77:2 197, 1980). The coding sequences for the heavy andlight chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced byrecombinant expression, it may be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A purification, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard techniques for the purification of proteins. Further, theantibodies of the present invention or fragments thereof may be fused toheterologous polypeptide sequences described herein or otherwise knownin the art to facilitate purification.

5.3.1 Antibody Conjugates

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to heterologous polypeptides (i.e., an unrelatedpolypeptide; or portion thereof, preferably at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90 or at least 100 amino acids of the polypeptide) togenerate fusion proteins. The fusion does not necessarily need to bedirect, but may occur through linker sequences. Antibodies fused orconjugated to heterologous polypeptides may also be used in in vitroimmunoassays and purification methods using methods known in the art.See e.g., PCT Publication No. WO 93/21232; EP 439,095; Naramura et al.,Immunol. Lett., 39:91-99, 1994; U.S. Pat. No. 5,474,981; Gillies et al.,PNAS, 89:1428-1432, 1992; and Fell et al., J. Immunol., 146:2446-2452,1991, which are incorporated herein by reference in their entireties.

Antibodies can be fused to marker sequences, such as a peptide tofacilitate purification. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., Proc. Natl. Acad. Sci. USA, 86:821-824, 1989, forinstance, hexa-histidine provides for convenient purification of thefusion protein. Other peptide tags useful for purification include, butare not limited to, the hemagglutinin “HA” tag, which corresponds to anepitope derived from the influenza hemagglutinin protein (Wilson et al.,Cell, 37:767 1984) and the “flag” tag (Knappik et al., Biotechniques,17(4):754-761, 1994).

The present invention also encompasses antibodies conjugated to adiagnostic or therapeutic agent or any other molecule for which in vivohalf-life is desired to be increased. The antibodies can be useddiagnostically to, for example, monitor the development or progressionof a disease, disorder or infection as part of a clinical testingprocedure to, e.g., determine the efficacy of a given treatment regimen.Detection can be facilitated by coupling the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, radioactive materials, positron emittingmetals, and nonradioactive paramagnetic metal ions. The detectablesubstance may be coupled or conjugated either directly to the antibodyor indirectly, through an intermediate (such as, for example, a linkerknown in the art) using techniques known in the art. See, for example,U.S. Pat. No. 4,741,900 for metal ions which can be conjugated toantibodies for use as diagnostics according to the present invention.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, f3-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ¹¹¹Inor ^(99m)Tc.

An antibody may be conjugated to a therapeutic moiety such as acytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agentor a radioactive element (e.g., alpha-emitters, gamma-emitters, etc.).Cytotoxins or cytotoxic agents include any agent that is detrimental tocells. Examples include paclitaxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, andanthramycin (AMC)), and anti-mitotic agents (e.g., vincristine andvinblastine).

Further, an antibody may be conjugated to a therapeutic agent or drugmoiety that modifies a given biological response. Therapeutic agents ordrug moieties are not to be construed as limited to classical chemicaltherapeutic agents. For example, the drug moiety may be a protein orpolypeptide possessing a desired biological activity. Such proteins mayinclude, for example, a toxin such as abrin, ricin A, pseudomonasexotoxin, or diphtheria toxin; a protein such as tumor necrosis factor,α-interferon (IFN-α), β-interferon (IFN-β), nerve growth factor (NGF),platelet derived growth factor (PDGF), tissue plasminogen activator(TPA), an apoptotic agent (e.g., TNF-α, TNF-β, AIM I as disclosed in PCTPublication No. WO 97/33899), AIM II (see, PCT Publication No. WO97/34911), Fas Ligand (Takahashi et al., J. Immunol., 6:1567-1574,1994), and VEGI (PCT Publication No. WO 99/23105), a thrombotic agent oran anti-angiogenic agent (e.g., angiostatin or endostatin); or abiological response modifier such as, for example, a lymphokine (e.g.,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), andgranulocyte colony stimulating factor (“G-CSF”)), or a growth factor(e.g., growth hormone (“GH”)).

Techniques for conjugating such therapeutic moieties to antibodies arewell known; see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), 1985, pp. 243-56, Alan R.Liss, Inc.); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), 1987, pp.623-53, Marcel Dekker, Inc.); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), 1985, pp.475-506); “Analysis, Results, And Future Prospective Of The TherapeuticUse Of Radiolabeled Antibody In Cancer Therapy”, in MonoclonalAntibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),1985, pp. 303-16, Academic Press; and Thorpe et al., Immunol.Recombinant expression vector., 62:119-58, 1982.

An antibody or fragment thereof, with or without a therapeutic moietyconjugated to it, administered alone or in combination with cytotoxicfactor(s) and/or cytokine(s) can be used as a therapeutic.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

5.4 Methods of Producing Fusion Proteins

Fusion proteins can be produced by standard recombinant DNA techniquesor by protein synthetic techniques, e.g., by use of a peptidesynthesizer. For example, a nucleic acid molecule encoding a fusionprotein can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed and reamplified to generate a chimeric genesequence (see, e.g., Current Protocols in Molecular Biology, Ausubel etal., eds., John Wiley & Sons, 1992). Moreover, a nucleic acid encoding abioactive molecule can be cloned into an expression vector containingthe Fc domain or a fragment thereof such that the bioactive molecule islinked in-frame to the constant domain or fragment thereof.

Methods for fusing or conjugating polypeptides to the constant regionsof antibodies are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603,5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181,5,908,626, 5,844,095, and 5,112,946; EP 307,434; EP 367,166; EP 394,827;PCT publications WO 91/06570, WO 96/04388, WO 96/22024, WO 97/34631, andWO 99/04813; Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88:10535-10539, 1991; Traunecker et al., Nature, 331:84-86, 1988; Zheng etal., J. Immunol., 154:5590-5600, 1995; and Vil et al., Proc. Natl. Acad.Sci. USA, 89:11337-11341, 1992, which are incorporated herein byreference in their entireties.

The nucleotide sequence encoding a bioactive molecule may be obtainedfrom any information available to those of skill in the art (e.g., fromGenbank, the literature, or by routine cloning), and the nucleotidesequence encoding a constant domain or a fragment thereof with increasedaffinity for the FcRn may be determined by sequence analysis of mutantsproduced using techniques described herein, or may be obtained fromGenbank or the literature. The nucleotide sequence coding for a fusionprotein can be inserted into an appropriate expression vector, i.e., avector which contains the necessary elements for the transcription andtranslation of the inserted protein-coding sequence. A variety ofhost-vector systems may be utilized in the present invention to expressthe protein-coding sequence. These include but are not limited tomammalian cell systems infected with virus (e.g., vaccinia virus,adenovirus, etc.); insect cell systems infected with virus (e.g.,baculovirus); microorganisms such as yeast containing yeast vectors; orbacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmidDNA. The expression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

The expression of a fusion protein may be controlled by any promoter orenhancer element known in the art. Promoters which may be used tocontrol the expression of the gene encoding fusion protein include, butare not limited to, the SV40 early promoter region (Bernoist andChambon, Nature, 290:304-310, 1981), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto, et al., Cell,22:787-797, 1980), the herpes thymidine kinase promoter (Wagner et al.,Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445, 1981), the regulatorysequences of the metallothionein gene (Brinster et al., Nature,296:39-42, 1982), the tetracycline (Tet) promoter (Gossen et al., Proc.Nat. Acad. Sci. USA, 89:5547-5551, 1995); prokaryotic expression vectorssuch as the β-lactamase promoter (VIIIa-Kamaroff, et al., Proc. Natl.Acad. Sci. U.S.A., 75:3727-3731, 1978), or the tac promoter (DeBoer, etal., Proc. Natl. Acad. Sci. U.S.A., 80:21-25, 1983; see also “Usefulproteins from recombinant bacteria” in Scientific American, 242:74-94,1980); plant expression vectors comprising the nopaline synthetasepromoter region (Herrera-Estrella et al., Nature, 303:209-213, 1983) orthe cauliflower mosaic virus 35S RNA promoter (Gardner, et al., Nucl.Acids Res., 9:2871, 1981), and the promoter of the photosynthetic enzymeribulose biphosphate carboxylase (Herrera-Estrella et al., Nature,310:115-120, 1984); promoter elements from yeast or other fungi such asthe Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK(phosphoglycerol kinase) promoter, alkaline phosphatase promoter, andthe following animal transcriptional control regions, which exhibittissue specificity and have been utilized in transgenic animals:elastase I gene control region which is active in pancreatic acinarcells (Swift et al., Cell 38:639-646, 1984; Ornitz et al., 50:399-409,Cold Spring Harbor Symp. Quant. Biol., 1986; MacDonald, Hepatology7:425-515, 1987); insulin gene control region which is active inpancreatic beta cells (Hanahan, Nature 315:115-122, 1985),immunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., Cell, 38:647-658, 1984; Adames et al., Nature318:533-538, 1985; Alexander et al., Mol. Cell. Biol., 7:1436-1444,1987), mouse mammary tumor virus control region which is active intesticular, breast, lymphoid and mast cells (Leder et al., Cell,45:485-495, 1986), albumin gene control region which is active in liver(Pinkert et al., Genes and Devel., 1:268-276, 1987), α-fetoprotein genecontrol region which is active in liver (Krumlauf et al., Mol. Cell.Biol., 5:1639-1648, 1985; Hammer et al., Science, 235:53-58, 1987; a1-antitrypsin gene control region which is active in the liver (Kelseyet al., Genes and Devel., 1:161-171, 1987), beta-globin gene controlregion which is active in myeloid cells (Mogram et al., Nature,315:338-340, 1985; Kollias et al., Cell, 46:89-94, 1986; myelin basicprotein gene control region which is active in oligodendrocyte cells inthe brain (Readhead et al., Cell, 48:703-712, 1987); myosin lightchain-2 gene control region which is active in skeletal muscle (Sani,Nature, 314:283-286, 1985); neuronal-specific enolase (NSE) which isactive in neuronal cells (Morelli et al., Gen. Virol., 80:571-83, 1999);brain-derived neurotrophic factor (BDNF) gene control region which isactive in neuronal cells (Tabuchi et al., Biochem. Biophysic. Res.Comprising., 253:818-823, 1998); glial fibrillary acidic protein (GFAP)promoter which is active in astrocytes (Gomes et al., Braz. J. Med.Biol. Res., 32(5):619-631, 1999; Morelli et al., Gen. Virol., 80:571-83,1999) and gonadotropic releasing hormone gene control region which isactive in the hypothalamus (Mason et al., Science, 234:1372-1378, 1986).

In a specific embodiment, the expression of a fusion protein isregulated by a constitutive promoter. In another embodiment, theexpression of a fusion protein is regulated by an inducible promoter. Inaccordance with these embodiments, the promoter may be a tissue-specificpromoter.

In a specific embodiment, a vector is used that comprises a promoteroperably linked to a fusion protein-encoding nucleic acid, one or moreorigins of replication, and, optionally, one or more selectable markers(e.g., an antibiotic resistance gene).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the fusion protein coding sequence may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts (e.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA, 81:355-359, 1984). Specificinitiation signals may also be required for efficient translation ofinserted fusion protein coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bitter et al., Methods inEnzymol., 153:516-544, 1987).

Expression vectors containing inserts of a gene encoding a fusionprotein can be identified by three general approaches: (a) nucleic acidhybridization, (b) presence or absence of “marker” gene functions, and(c) expression of inserted sequences. In the first approach, thepresence of a gene encoding a fusion protein in an expression vector canbe detected by nucleic acid hybridization using probes comprisingsequences that are homologous to an inserted gene encoding the fusionprotein. In the second approach, the recombinant vector/host system canbe identified and selected based upon the presence or absence of certain“marker” gene functions (e.g., thymidine kinase activity, resistance toantibiotics, transformation phenotype, occlusion body formation inbaculovirus, etc.) caused by the insertion of a nucleotide sequenceencoding a fusion protein in the vector. For example, if the nucleotidesequence encoding the fusion protein is inserted within the marker genesequence of the vector, recombinants containing the gene encoding thefusion protein insert can be identified by the absence of the markergene function. In the third approach, recombinant expression vectors canbe identified by assaying the gene product (i.e., fusion protein)expressed by the recombinant. Such assays can be based, for example, onthe physical or functional properties of the fusion protein in in vitroassay systems, e.g., binding with anti-bioactive molecule antibody.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered fusion protein may becontrolled. Furthermore, different host cells have characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification (e.g., glycosylation, phosphorylation ofproteins). Appropriate cell lines or host systems can be chosen toensure the desired modification and processing of the foreign proteinexpressed. For example, expression in a bacterial system will produce anunglycosylated product and expression in yeast will produce aglycosylated product. Eukaryotic host cells which possess the cellularmachinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product may be used. Suchmammalian host cells include but are not limited to CHO, VERY, BHK,HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, neuronal cell linessuch as, for example, SK-N-AS, SK-N-FI, SK-N-DZ human neuroblastomas(Sugimoto et al., J. Natl. Cancer Inst., 73: 51-57, 1984), SK-N-SH humanneuroblastoma (Biochim. Biophys. Acta, 704: 450-460, 1982), Daoy humancerebellar medulloblastoma (He et al., Cancer Res., 52: 1144-1148, 1992)DBTRG-05MG glioblastoma cells (Kruse et al., 1992, In Vitro Cell. Dev.Biol., 28A:609-614, 1992), IMR-32 human neuroblastoma (Cancer Res., 30:2110-2118, 1970), 1321N1 human astrocytoma (Proc. Nall Acad. Sci. USA,74: 4816, 1997), MOG-G-CCM human astrocytoma (Br. J. Cancer, 49: 269,1984), U87MG human glioblastoma-astrocytoma (Acta Pathol. Microbiol.Scand., 74: 465-486, 1968), A172 human glioblastoma (Olopade et al.,Cancer Res., 52: 2523-2529, 1992), C6 rat glioma cells (Benda et al.,Science, 161: 370-371, 1968), Neuro-2a mouse neuroblastoma (Proc. Natl.Acad. Sci. USA, 65: 129-136, 1970), NB41A3 mouse neuroblastoma (Proc.Natl. Acad. Sci. USA, 48: 1184-1190, 1962), SCP sheep choroid plexus(Bolin et al., J. Virol. Methods, 48: 211-221, 1994), G355-5, PG-4 Catnormal astrocyte (Haapala et al., J. Virol., 53: 827-833, 1985), Mpfferret brain (Trowbridge et al., In Vitro, 18: 952-960, 1982), andnormal cell lines such as, for example, CTX TNA2 rat normal cortex brain(Radany et al., Proc. Natl. Acad. Sci. USA, 89: 6467-6471, 1992) suchas, for example, CRL7030 and Hs578Bst. Furthermore, differentvector/host expression systems may effect processing reactions todifferent degrees.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe fusion protein may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched medium, and then areswitched to a selective medium. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into their chromosomes and grow to form foci whichin turn can be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines that express thedifferentially expressed or pathway gene protein. Such engineered celllines may be particularly useful in screening and evaluation ofcompounds that affect the endogenous activity of the differentiallyexpressed or pathway gene protein.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., Cell, 11:223,1997), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA, 48:2026, 1962), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell, 22:817, 1980) genescan be employed in tk−, hgprt− or aprt− cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler, et al., Natl.Acad. Sci. USA, 77:3567, 1980; O'Hare, et al., Proc. Natl. Acad. Sci.USA, 78:1527, 1981); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072, 1981); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin, etal., J. Mol. Biol., 150:1, 1981); and hygro, which confers resistance tohygromycin (Santerre, et al., Gene, 30:147, 1984) genes.

Once a fusion protein of the invention has been produced by recombinantexpression, it may be purified by any method known in the art forpurification of a protein, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins.

5.5 Prophylactic and Therapeutic Uses of Antibodies

The present invention encompasses antibody-based therapies which involveadministering antibodies to an animal, preferably a mammal, and mostpreferably a human, for preventing, treating, or ameliorating symptomsassociated with a disease, disorder, or infection. Prophylactic andtherapeutic compounds of the invention include, but are not limited to,antibodies and nucleic acids encoding antibodies. Antibodies may beprovided in pharmaceutically acceptable compositions as known in the artor as described herein.

Antibodies of the present invention that function as antagonists of adisease, disorder, or infection can be administered to an animal,preferably a mammal and most preferably a human, to treat, prevent orameliorate one or more symptoms associated with the disease, disorder,or infection. For example, antibodies which disrupt or prevent theinteraction between a viral antigen and its host cell receptor may beadministered to an animal, preferably a mammal and most preferably ahuman, to treat, prevent or ameliorate one or more symptoms associatedwith a viral infection.

In a specific embodiment, an antibody or fragment thereof prevents aviral or bacterial antigen from binding to its host cell receptor by atleast 99%, at least 95%, at least 90%, at least 85%, at least 80%, atleast 75%, at least 70%, at least 60%, at least 50%, at least 45%, atleast 40%, at least 45%, at least 35%, at least 30%, at least 25%, atleast 20%, or at least 10% relative to antigen binding to its host cellreceptor in the absence of said antibodies. In another embodiment, acombination of antibodies prevent a viral or bacterial antigen frombinding to its host cell receptor by at least 99%, at least 95%, atleast 90%, at least 85%, at least 80%, at least 75%, at least 70%, atleast 60%, at least 50%, at least 45%, at least 40%, at least 45%, atleast 35%, at least 30%, at least 25%, at least 20%, or at least 10%relative to antigen binding to its host cell receptor in the absence ofsaid antibodies. In a preferred embodiment, the antibody is used totreat or prevent RSV infection

Antibodies which do not prevent a viral or bacterial antigen frombinding its host cell receptor but inhibit or downregulate viral orbacterial replication can also be administered to an animal to treat,prevent or ameliorate one or more symptoms associated with a viral orbacterial infection. The ability of an antibody to inhibit ordownregulate viral or bacterial replication may be determined bytechniques described herein or otherwise known in the art. For example,the inhibition or downregulation of viral replication can be determinedby detecting the viral titer in the animal.

In a specific embodiment, an antibody inhibits or downregulates viral orbacterial replication by at least 99%, at least 95%, at least 90%, atleast 85%, at least 80%, at least 75%, at least 70%, at least 60%, atleast 50%, at least 45%, at least 40%, at least 45%, at least 35%, atleast 30%, at least 25%, at least 20%, or at least 10% relative to viralor bacterial replication in absence of said antibody. In anotherembodiment, a combination of antibodies inhibit or downregulate viral orbacterial replication by at least 99%, at least 95%, at least 90%, atleast 85%, at least 80%, at least 75%, at least 70%, at least 60%, atleast 50%, at least 45%, at least 40%, at least 45%, at least 35%, atleast 30%, at least 25%, at least 20%, or at least 10% relative to viralor bacterial replication in absence of said antibodies.

Antibodies can also be used to prevent, inhibit or reduce the growth ormetastasis of cancerous cells. In a specific embodiment, an antibodyinhibits or reduces the growth or metastasis of cancerous cells by atleast 99%, at least 95%, at least 90%, at least 85%, at least 80%, atleast 75%, at least 70%, at least 60%, at least 50%, at least 45%, atleast 40%, at least 45%, at least 35%, at least 30%, at least 25%, atleast 20%, or at least 10% relative to the growth or metastasis inabsence of said antibody. In another embodiment, a combination ofantibodies inhibits or reduces the growth or metastasis of cancer by atleast 99%, at least 95%, at least 90%, at least 85%, at least 80%, atleast 75%, at least 70%, at least 60%, at least 50%, at least 45%, atleast 40%, at least 45%, at least 35%, at least 30%, at least 25%, atleast 20%, or at least 10% relative to the growth or metastasis inabsence of said antibodies. Examples of cancers include, but are notlimited to, leukemia (e.g., acute leukemia such as acute lymphocyticleukemia and acute myelocytic leukemia), neoplasms, tumors (e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma), heavy chain disease, metastases, or any disease ordisorder characterized by uncontrolled cell growth.

Antibodies can also be used to reduce the inflammation experienced byanimals, particularly mammals, with inflammatory disorders. In aspecific embodiment, an antibody reduces the inflammation in an animalby at least 99%, at least 95%, at least 90%, at least 85%, at least 80%,at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, atleast 40%, at least 45%, at least 35%, at least 30%, at least 25%, atleast 20%, or at least 10% relative to the inflammation in an animal inthe not administered said antibody. In another embodiment, a combinationof antibodies reduce the inflammation in an animal by at least 99%, atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 60%, at least 50%, at least 45%, at least 40%, atleast 45%, at least 35%, at least 30%, at least 25%, at least 20%, or atleast 10% relative to the inflammation in an animal in not administeredsaid antibodies. Examples of inflammatory disorders include, but are notlimited to, rheumatoid arthritis, spondyloarthropathies, inflammatorybowel disease and asthma.

In certain embodiments, the antibody used for treatment of inflammation(or cancer) is a modified anti-α_(v)β₃ antibody, preferably a Vitaxinantibody (see, PCT publications WO 98/33919 and WO 00/78815, both byHuse et al., and both of which are incorporated by reference herein intheir entireties).

Antibodies can also be used to prevent the rejection of transplants.Antibodies can also be used to prevent clot formation. Further,antibodies that function as agonists of the immune response can also beadministered to an animal, preferably a mammal, and most preferably ahuman, to treat, prevent or ameliorate one or more symptoms associatedwith the disease, disorder, or infection.

One or more antibodies that immunospecifically bind to one or moreantigens may be used locally or systemically in the body as atherapeutic. The antibodies of this invention may also be advantageouslyutilized in combination with other monoclonal or chimeric antibodies, orwith lymphokines or hematopoietic growth factors (such as, e.g., IL-2,IL-3 and IL-7), which, for example, serve to increase the number oractivity of effector cells which interact with the antibodies. Theantibodies of this invention may also be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), which, for example, serve to increase the immune response.The antibodies of this invention may also be advantageously utilized incombination with one or more drugs used to treat a disease, disorder, orinfection such as, for example anti-cancer agents, anti-inflammatoryagents or anti-viral agents. Examples of anti-cancer agents include, butare not limited to, isplatin, ifosfamide, paclitaxel, taxanes,topoisomerase I inhibitors (e.g., CPT-11, topotecan, 9-AC, and GG-211),gemcitabine, vinorelbine, oxaliplatin, 5-fluorouracil (5-FU),leucovorin, vinorelbine, temodal, and taxol. Examples of anti-viralagents include, but are not limited to, cytokines (e.g., IFN-α, IFN-β,IFN-γ), inhibitors of reverse transcriptase (e.g., AZT, 3TC, D4T, ddC,ddI, d4T, 3TC, adefovir, efavirenz, delavirdine, nevirapine, abacavir,and other dideoxynucleosides or dideoxyfluoronucleosides), inhibitors ofviral mRNA capping, such as ribavirin, inhibitors of proteases such HIVprotease inhibitors (e.g., amprenavir, indinavir, nelfinavir, ritonavir,and saquinavir), amphotericin B, castanospermine as an inhibitor ofglycoprotein processing, inhibitors of neuraminidase such as influenzavirus neuraminidase inhibitors (e.g., zanamivir and oseltamivir),topoisomerase I inhibitors (e.g., camptothecins and analogs thereof),amantadine, and rimantadine. Examples of anti-inflammatory agentsinclude, but are not limited to, nonsteroidal anti-inflammatory drugssuch as COX-2 inhibitors (e.g., meloxicam, celecoxib, rofecoxib,flosulide, and SC-58635, and MK-966), ibuprofen and indomethacin, andsteroids (e.g., deflazacort, dexamethasone and methylprednisolone).

In a specific embodiment, antibodies administered to an animal are of aspecies origin or species reactivity that is the same species as that ofthe animal. Thus, in a preferred embodiment, human or humanizedantibodies, or nucleic acids encoding human or human, are administeredto a human patient for therapy or prophylaxis.

In preferred embodiments, immunoglobulins having extended in vivohalf-lives are used in passive immunotherapy (for either therapy orprophylaxis). Because of the extended half-life, passive immunotherapyor prophylaxis can be accomplished using lower doses and/or lessfrequent administration of the therapeutic resulting in fewer sideeffects, better patient compliance, less costly therapy/prophylaxis,etc. In a preferred embodiment, the therapeutic/prophylactic is anantibody that binds RSV, for example, SYNAGIS® or other anti-RSVantibody. Such anti-RSV antibodies, and methods of administration aredisclosed in U.S. patent application Ser. Nos. 09/724,396 and09/724,531, both entitled “Methods of Administering/Dosing Anti-RSVAntibodies For Prophylaxis and Treatment,” both by Young et al., bothfiled Nov. 28, 2000, and continuation-in-part applications of theseapplication Ser. Nos. 09/996,265 and 09/996,288, respectively (attorneydocket Nos. 10271-048 and 10271-047, respectively), both filed Nov. 28,2001, also entitled “Methods of Administering/Dosing Anti-RSV Antibodiesfor Prophylaxis and Treatment,” by Young et al., all which areincorporated by reference herein in their entireties. Also included arethe anti-RSV antibodies described in Section 5.1, supra.

In a specific embodiment, fusion proteins administered to an animal areof a species origin or species reactivity that is the same species asthat of the animal. Thus, in a preferred embodiment, human fusionproteins or nucleic acids encoding human fusion proteins, areadministered to a human subject for therapy or prophylaxis.

5.6 Prophylactic and Therapeutic Uses of Fusion Proteins and ConjugatedMolecules

The present invention encompasses fusion protein-based and conjugatedmolecule-based therapies which involve administering fusion proteins orconjugated molecules to an animal, preferably a mammal and mostpreferably a human, for preventing, treating, or ameliorating symptomsassociated with a disease, disorder, or infection. Prophylactic andtherapeutic compounds of the invention include, but are not limited to,fusion proteins and nucleic acids encoding fusion proteins andconjugated molecules. Fusion proteins and conjugated molecules may beprovided in pharmaceutically acceptable compositions as known in the artor as described herein.

Fusion proteins and conjugated molecules of the present invention thatfunction as antagonists of a disease, disorder, or infection can beadministered to an animal, preferably a mammal, and most preferably ahuman, to treat, prevent or ameliorate one or more symptoms associatedwith the disease, disorder, or infection. Further, fusion proteins andconjugated molecules of the present invention that function as agonistsof the immune response may be administered to an animal, preferably amammal, and most preferably a human, to treat, prevent or ameliorate oneor more symptoms associated with the disease, disorder, or infection.

One or more fusion proteins and conjugated molecules may be used locallyor systemically in the body as a therapeutic. The fusion proteins andconjugated molecules of this invention may also be advantageouslyutilized in combination with monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), which, for example, serve to increase the number or activityof effector cells which interact with the antibodies. The fusionproteins and conjugated molecules of this invention may also beadvantageously utilized in combination with monoclonal or chimericantibodies, or with lymphokines or hematopoietic growth factors (suchas, e.g., IL-2, IL-3 and IL-7), which, for example, serve to increasethe immune response. The fusion proteins and conjugated molecules ofthis invention may also be advantageously utilized in combination withone or more drugs used to treat a disease, disorder, or infection suchas, for example anti-cancer agents, anti-inflammatory agents oranti-viral agents. Examples of anti-cancer agents include, but are notlimited to, isplatin, ifosfamide, paclitaxel, taxanes, topoisomerase Iinhibitors (e.g., CPT-11, topotecan, 9-AC, and GG-211), gemcitabine,vinorelbine, oxaliplatin, 5-fluorouracil (5-FU), leucovorin,vinorelbine, temodal, and taxol. Examples of anti-viral agents include,but are not limited to, cytokines (e.g., IFN-α, IFN-β, IFN-γ),inhibitors of reverse transcriptase (e.g., AZT, 3TC, D4T, ddC, ddI, d4T,3TC, adefovir, efavirenz, delavirdine, nevirapine, abacavir, and otherdideoxynucleosides or dideoxyfluoronucleosides), inhibitors of viralmRNA capping, such as ribavirin, inhibitors of proteases such HIVprotease inhibitors (e.g., amprenavir, indinavir, nelfinavir, ritonavir,and saquinavir), amphotericin B, castanospermine as an inhibitor ofglycoprotein processing, inhibitors of neuraminidase such as influenzavirus neuraminidase inhibitors (e.g., zanamivir and oseltamivir),topoisomerase I inhibitors (e.g., camptothecins and analogs thereof),amantadine, and rimantadine. Examples of anti-inflammatory agentsinclude, but are not limited to, nonsteroidal anti-inflammatory drugssuch as COX-2 inhibitors (e.g., meloxicam, celecoxib, rofecoxib,flosulide, and SC-58635, and MK-966), ibuprofen and indomethacin, andsteroids (e.g., deflazacort, dexamethasone and methylprednisolone).

5.7 Administration of Antibodies or Fusion Proteins

The invention provides methods of treatment, prophylaxis, andamelioration of one or more symptoms associated with a disease, disorderor infection by administrating to a subject of an effective amount of anantibody of the invention, or pharmaceutical composition comprising anantibody of the invention. The invention also provides methods oftreatment, prophylaxis, and amelioration of one or more symptomsassociated with a disease, disorder or infection by administering to asubject an effective amount of a fusion protein or conjugated moleculeof the invention, or a pharmaceutical composition comprising a fusionprotein or conjugated molecules of the invention. In a preferred aspect,an antibody or fusion protein or conjugated molecule, is substantiallypurified (i.e., substantially free from substances that limit its effector produce undesired side-effects). In a specific embodiment, thesubject is an animal, preferably a mammal such as non-primate (e.g.,cows, pigs, horses, cats, dogs, rats etc.) and a primate (e.g., monkeysuch as a cynomolgous monkey and a human). In a preferred embodiment,the subject is a human.

Various delivery systems are known and can be used to administer anantibody or fusion protein or conjugated molecule of the invention,e.g., encapsulation in liposomes, microparticles, microcapsules,recombinant cells capable of expressing the antibody or fusion protein,receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem.,262:4429-4432, 1987), construction of a nucleic acid as part of aretroviral or other vector, etc. Methods of administering an antibody, afusion protein or conjugated molecule, or pharmaceutical compositioninclude, but are not limited to, parenteral administration (e.g.,intradermal, intramuscular, intraperitoneal, intravenous andsubcutaneous), epidural, and mucosal (e.g., intranasal and oral routes).In a specific embodiment, antibodies, fusion proteins, conjugatedmolecules, or pharmaceutical compositions are administeredintramuscularly, intravenously, or subcutaneously. The compositions maybe administered by any convenient route, for example by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and maybe administered together with other biologically active agents.Administration can be systemic or local. In addition, pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent. See, e.g., U.S.Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064;5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903, eachof which is incorporated herein by reference in its entirety. In apreferred embodiment, an antibody, a fusion protein, conjugatedmolecules, or a pharmaceutical composition is administered usingAlkermes AIR™ pulmonary drug delivery technology (Alkermes, Inc.,Cambridge, Mass.).

The invention also provides that an antibody, a fusion protein, orconjugated molecule is packaged in a hermetically sealed container suchas an ampoule or sachette indicating the quantity of antibody, fusionprotein, or conjugated molecule. In one embodiment, the antibody, fusionprotein, or conjugated molecule is supplied as a dry sterilizedlyophilized powder or water free concentrate in a hermetically sealedcontainer and can be reconstituted, e.g., with water or saline to theappropriate concentration for administration to a subject. Preferably,the antibody, fusion protein, or conjugated molecule is supplied as adry sterile lyophilized powder in a hermetically sealed container at aunit dosage of at least 5 mg, more preferably at least 10 mg, at least15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg,or at least 75 mg. The lyophilized antibody, fusion protein, orconjugated molecule should be stored at between 2 and 8° C. in itsoriginal container and the antibody, fusion protein, or conjugatedmolecules should be administered within 12 hours, preferably within 6hours, within 5 hours, within 3 hours, or within 1 hour after beingreconstituted. In an alternative embodiment, an antibody, fusionprotein, or conjugated molecule is supplied in liquid form in ahermetically sealed container indicating the quantity and concentrationof the antibody, fusion protein, or conjugated molecule. Preferably, theliquid form of the antibody, fusion protein, or conjugated molecule issupplied in a hermetically sealed container at least 1 mg/ml, morepreferably at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, atleast 10 mg/ml, at least 15 mg/kg, or at least 25 mg/ml.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion, by injection, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. Preferably,when administering an antibody or a fusion protein, care must be takento use materials to which the antibody or the fusion protein does notabsorb.

In another embodiment, the composition can be delivered in a vesicle, inparticular a liposome (see Langer, Science, 249:1527-1533, 1990; Treatet al., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In yet another embodiment, the composition can be delivered in acontrolled release or sustained release system. Any technique known toone of skill in the art can be used to produce sustained releaseformulations comprising one or more antibodies, or one or more fusionproteins. See, e.g., U.S. Pat. No. 4,526,938; PCT publication WO91/05548; PCT publication WO 96/20698; Ning et al., “IntratumoralRadioimmunotheraphy of a Human Colon Cancer Xenograft Using aSustained-Release Gel,” Radiotherapy & Oncology, 39:179-189, 1996; Songet al., “Antibody Mediated Lung Targeting of Long-CirculatingEmulsions,” PDA Journal of Pharmaceutical Science & Technology,50:372-397, 1995; Cleek et al., “Biodegradable Polymeric Carriers for abFGF Antibody for Cardiovascular Application,” Pro. Intl. Symp. Control.Rel. Bioact. Mater., 24:853-854, 1997; and Lam et al.,“Microencapsulation of Recombinant Humanized Monoclonal Antibody forLocal Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater.,24:759-760, 1997, each of which is incorporated herein by reference inits entirety. In one embodiment, a pump may be used in a controlledrelease system (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng.,14:20, 1987; Buchwald et al., Surgery, 88:507, 1980; and Saudek et al.,N. Engl. J. Med., 321:574, 1989). In another embodiment, polymericmaterials can be used to achieve controlled release of antibodies orfusion proteins (see e.g., Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol.Sci. Rev. Macromol. Chem., 23:61, 1983; see also Levy et al., Science,228:190, 1985; During et al., Ann. Neurol., 25:351, 1989; Howard et al.,J. Neurosurg., 7 1:105, 1989); U.S. Pat. No. 5,679,377; U.S. Pat. No.5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat.No. 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No.WO 99/20253). In yet another embodiment, a controlled release system canbe placed in proximity of the therapeutic target (e.g., the lungs), thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

Other controlled release systems are discussed in the review by Langer,Science, 249:1527-1533, 1990).

In a specific embodiment where the composition of the invention is anucleic acid encoding an antibody or fusion protein, the nucleic acidcan be administered in vivo to promote expression of its encodedantibody or fusion protein, by constructing it as part of an appropriatenucleic acid expression vector and administering it so that it becomesintracellular, e.g., by use of a retroviral vector (see U.S. Pat. No.4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun; Biolistic, Dupont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-like peptide which is known toenter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA,88:1864-1868, 1991), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a prophylactically or therapeutically effectiveamount of an antibody, fusion protein or conjugated molecule, and apharmaceutically acceptable carrier. In a specific embodiment, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent, adjuvant(e.g., Freund's complete and incomplete, mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful adjuvants for humanssuch as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum),excipient, or vehicle with which the therapeutic is administered. Suchpharmaceutical carriers can be sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.Water is a preferred carrier when the pharmaceutical composition isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. The composition, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa prophylactically or therapeutically effective amount of the antibodyor fragment thereof, or fusion protein or conjugated molecule,preferably in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection.

Generally, the ingredients of compositions of the invention are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compositions of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the composition of the invention which will be effectivein the treatment, prevention or amelioration of one or more symptomsassociated with a disease, disorder, or infection can be determined bystandard clinical techniques. The precise dose to be employed in theformulation will depend on the route of administration, the age of thesubject, and the seriousness of the disease, disorder, or infection, andshould be decided according to the judgment of the practitioner and eachpatient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model (e.g., thecotton rat or Cynomolgous monkey) test systems.

For fusion proteins, the therapeutically or prophylactically effectivedosage administered to a subject ranges from about 0.001 to 50 mg/kgbody weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. For antibodies, the therapeutically orprophylactically effective dosage administered to a subject is typically0.1 mg/kg to 200 mg/kg of the subject's body weight. Preferably, thedosage administered to a subject is between 0.1 mg/kg and 20 mg/kg ofthe subject's body weight and more preferably the dosage administered toa subject is between 1 mg/kg to 10 mg/kg of the subject's body weight.The dosage will, however, depend upon the extent to which the in vivohalf-life of the molecule has been increased Generally, human antibodiesand human fusion proteins have longer half-lives within the human bodythan antibodies of fusion proteins from other species due to the immuneresponse to the foreign polypeptides. Thus, lower dosages of humanantibodies or human fusion proteins and less frequent administration isoften possible. Further, the dosage and frequency of administration ofantibodies, fusion proteins, or conjugated molecules may be reduced alsoby enhancing uptake and tissue penetration (e.g., into the lung) of theantibodies or fusion proteins by modifications such as, for example,lipidation.

Treatment of a subject with a therapeutically or prophylacticallyeffective amount of an antibody, fusion protein, or conjugated moleculecan include a single treatment or, preferably, can include a series oftreatments. In a preferred example, a subject is treated with anantibody, fusion protein, or conjugated molecule in the range of betweenabout 0.1 to 30 mg/kg body weight, one time per week for between about 1to 10 weeks, preferably between 2 to 8 weeks, more preferably betweenabout 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.In other embodiments, the pharmaceutical composition of the invention isadministered once a day, twice a day, or three times a day. In otherembodiments, the pharmaceutical composition is administered once a week,twice a week, once every two weeks, once a month, once every six weeks,once every two months, twice a year or once per year. It will also beappreciated that the effective dosage of the antibody, fusion protein,or conjugated molecule used for treatment may increase or decrease overthe course of a particular treatment.

5.7.1 Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or fusion proteins, are administered to treat, prevent orameliorate one or more symptoms associated with a disease, disorder, orinfection, by way of gene therapy. Gene therapy refers to therapyperformed by the administration to a subject of an expressed orexpressible nucleic acid. In this embodiment of the invention, thenucleic acids produce their encoded antibody or fusion protein thatmediates a therapeutic or prophylactic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy, 12:488-505, 1993; Wu and Wu, Biotherapy,3:87-95, 1991; Tolstoshev, Ann. Rev. Pharmacol. Toxicol., 32:573-596,1993; Mulligan, Science, 260:926-932, 1993; and Morgan and Anderson,Ann. Rev. biochem. 62:191-217, 1993; TIBTECH 11(5):155-215, 1993.Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, a composition of the invention comprises nucleicacids encoding an antibody, said nucleic acids being part of anexpression vector that expresses the antibody in a suitable host. Inparticular, such nucleic acids have promoters, preferably heterologouspromoters, operably linked to the antibody coding region, said promoterbeing inducible or constitutive, and, optionally, tissue-specific. Inanother particular embodiment, nucleic acid molecules are used in whichthe antibody coding sequences and any other desired sequences areflanked by regions that promote homologous recombination at a desiredsite in the genome, thus providing for intrachromosomal expression ofthe antibody encoding nucleic acids (Koller and Smithies, Proc. Natl.Acad. Sci. USA, 86:8932-8935, 1989; and Zijlstra et al., Nature,342:435-438, 1989).

In another preferred aspect, a composition of the invention comprisesnucleic acids encoding a fusion protein, said nucleic acids being a partof an expression vector that expression the fusion protein in a suitablehost. In particular, such nucleic acids have promoters, preferablyheterologous promoters, operably linked to the coding region of a fusionprotein, said promoter being inducible or constitutive, and optionally,tissue-specific. In another particular embodiment, nucleic acidmolecules are used in which the coding sequence of the fusion proteinand any other desired sequences are flanked by regions that promotehomologous recombination at a desired site in the genome, thus providingfor intrachromosomal expression of the fusion protein encoding nucleicacids.

Delivery of the nucleic acids into a subject may be either direct, inwhich case the subject is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the subject. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretroviral or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987)(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316; WO 93/14188; WO 93/20221). Alternatively, the nucleic acid canbe introduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA, 86:8932-8935, 1989; and Zijlstra et al., Nature,342:435-438, 1989).

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding an antibody or a fusion protein are used. Forexample, a retroviral vector can be used (see Miller et al., Meth.Enzymol., 217:581-599, 1993). These retroviral vectors contain thecomponents necessary for the correct packaging of the viral genome andintegration into the host cell DNA. The nucleic acid sequences encodingthe antibody or a fusion protein to be used in gene therapy are clonedinto one or more vectors, which facilitates delivery of the nucleotidesequence into a subject. More detail about retroviral vectors can befound in Boesen et al., Biotherapy, 6:291-302, 1994, which describes theuse of a retroviral vector to deliver the mdr 1 gene to hematopoieticstem cells in order to make the stem cells more resistant tochemotherapy. Other references illustrating the use of retroviralvectors in gene therapy are: Clowes et al., J. Clin. Invest.,93:644-651, 1994; Klein et al., Blood 83:1467-1473, 1994; Salmons andGunzberg, Human Gene Therapy, 4:129-141, 1993; and Grossman and Wilson,Curr. Opin. in Genetics and Devel., 3:110-114, 1993.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development, 3:499-503, 1993, present a reviewof adenovirus-based gene therapy. Bout et al., Human Gene Therapy,5:3-10, 1994, demonstrated the use of adenovirus vectors to transfergenes to the respiratory epithelia of rhesus monkeys. Other instances ofthe use of adenoviruses in gene therapy can be found in Rosenfeld etal., Science, 252:431-434, 1991; Rosenfeld et al., Cell, 68:143-155,1992; Mastrangeli et al., J. Clin. Invest., 91:225-234, 1993; PCTPublication WO 94/12649; and Wang et al., Gene Therapy, 2:775-783, 1995.In a preferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (see, e.g., Walsh et al., Proc. Soc. Exp. Biol. Med.,204:289-300, 1993, and U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a subject.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcellmediated gene transfer, spheroplast fusion, etc.Numerous techniques are known in the art for the introduction of foreigngenes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.,217:599-618, 1993; Cohen et al., Meth. Enzymol., 217:618-644, 1993; andClin. Pharma. Ther., 29:69-92, 1985) and may be used in accordance withthe present invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a subject by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the subject.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody or a fusion protein areintroduced into the cells such that they are expressible by the cells ortheir progeny, and the recombinant cells are then administered in vivofor therapeutic effect. In a specific embodiment, stem or progenitorcells are used. Any stem and/or progenitor cells which can be isolatedand maintained in vitro can potentially be used in accordance with thisembodiment of the present invention (see e.g., PCT Publication WO94/08598; Stemple and Anderson, Cell, 7 1:973-985, 1992; Rheinwald,Meth. Cell Bio., 21A:229, 1980; and Pittelkow and Scott, Mayo ClinicProc., 61:771, 1986).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

5.8 Characterization and Demonstration of Therapeutic or ProphylacticUtility

Antibodies, fusion proteins, and conjugated molecules of the presentinvention may be characterized in a variety of ways. In particular,antibodies of the invention may be assayed for the ability toimmunospecifically bind to an antigen. Such an assay may be performed insolution (e.g., Houghten, Bio/Techniques, 13:412-421, 1992), on beads(Lam, Nature, 354:82-84, 1991, on chips (Fodor, Nature, 364:555-556,1993), on bacteria (U.S. Pat. No. 5,223,409), on spores (U.S. Pat. Nos.5,571,698; 5,403,484; and 5,223,409), on plasmids (Cull et al., Proc.Natl. Acad. Sci. USA, 89:1865-1869, 1992) or on phage (Scott and Smith,Science, 249:386-390, 1990; Devlin, Science, 249:404-406, 1990; Cwirlaet al., Proc. Natl. Acad. Sci. USA, 87:6378-6382, 1990; and Felici, J.Mol. Biol., 222:301-310, 1991) (each of these references is incorporatedherein in its entirety by reference). Antibodies that have beenidentified to immunospecifically bind to an antigen or a fragmentthereof can then be assayed for their specificity affinity for theantigen.

The antibodies of the invention or fragments thereof may be assayed forimmunospecific binding to an antigen and cross-reactivity with otherantigens by any method known in the art. Immunoassays which can be usedto analyze immunospecific binding and cross-reactivity include, but arenot limited to, competitive and non-competitive assay systems usingtechniques such as western blots, radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1 to 4 hours) at 40° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 40° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., ³²P or ¹²⁵I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., ³H or ¹²⁵I) withthe antibody of interest in the presence of increasing amounts ofunlabeled antigen, and the detection of the antibody bound to thelabeled antigen. The affinity of the antibody of the present inventionor a fragment thereof for the antigen and the binding off-rates can bedetermined from the saturation data by scatchard analysis. Competitionwith a second antibody can also be determined using radioimmunoassays.In this case, the antigen is incubated with an antibody of the presentinvention or a fragment thereof conjugated to a labeled compound (e.g.,³H or ¹²⁵I) in the presence of increasing amounts of an unlabeled secondantibody.

In a preferred embodiment, BIAcore kinetic analysis is used to determinethe binding on and off rates of antibodies to an antigen. BIAcorekinetic analysis comprises analyzing the binding and dissociation of anantigen from chips with immobilized antibodies on their surface (see theExample section infra).

The antibodies of the invention as well as fusion proteins andconjugated molecules can also be assayed for their ability to inhibitthe binding of an antigen to its host cell receptor using techniquesknown to those of skill in the art. For example, cells expressing thereceptor for a viral antigen can be contacted with virus in the presenceor absence of an antibody and the ability of the antibody to inhibitviral antigen's binding can measured by, for example, flow cytometry ora scintillation counter. The antigen or the antibody can be labeled witha detectable compound such as a radioactive label (e.g., ³²P, ³⁵S, and¹²⁵I) or a fluorescent label (e.g., fluorescein isothiocyanate,rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehydeand fluorescamine) to enable detection of an interaction between theantigen and its host cell receptor. Alternatively, the ability ofantibodies to inhibit an antigen from binding to its receptor can bedetermined in cell-free assays. For example, virus or a viral antigen(e.g., RSV F glycoprotein) can be contacted in a cell-free assay with anantibody and the ability of the antibody to inhibit the virus or theviral antigen from binding to its host cell receptor can be determined.Preferably, the antibody is immobilized on a solid support and theantigen is labeled with a detectable compound. Alternatively, theantigen is immobilized on a solid support and the antibody is labeledwith a detectable compound. The antigen may be partially or completelypurified (e.g., partially or completely free of other polypeptides) orpart of a cell lysate. Further, the antigen may be a fusion proteincomprising the viral antigen and a domain such asglutathionine-S-transferase. Alternatively, an antigen can bebiotinylated using techniques well known to those of skill in the art(e.g., biotinylation kit, Pierce Chemicals; Rockford, Ill.).

The antibodies, fusion proteins, and conjugated molecules of theinvention can also be assayed for their ability to inhibit ordownregulate viral or bacterial replication using techniques known tothose of skill in the art. For example, viral replication can be assayedby a plaque assay such as described, e.g., by Johnson et al., Journal ofInfectious Diseases, 176:1215-1224, 1997. The antibodies, fusionproteins, and conjugated molecules of the invention of the invention canalso be assayed for their ability to inhibit or downregulate theexpression of viral or bacterial polypeptides. Techniques known to thoseof skill in the art, including, but not limited to, Western blotanalysis, Northern blot analysis, and RT-PCR, can be used to measure theexpression of viral or bacterial polypeptides. Further, the antibodies,fusion proteins, and conjugated molecules of the invention of theinvention can be assayed for their ability to prevent the formation ofsyncytia.

The antibodies, fusion proteins, conjugated molecules, and compositionsof the invention are preferably tested in vitro, and then in vivo forthe desired therapeutic or prophylactic activity, prior to use inhumans. For example, in vitro assays which can be used to determinewhether administration of a specific antibody, a specific fusionprotein, a specific conjugated molecule, or a composition of the presentinvention is indicated, include in vitro cell culture assays in which asubject tissue sample is grown in culture, and exposed to or otherwiseadministered an antibody, a fusion protein, conjugated molecule, orcomposition of the present invention, and the effect of such anantibody, a fusion protein, conjugated molecule, or a composition of thepresent invention upon the tissue sample is observed. In variousspecific embodiments, in vitro assays can be carried out withrepresentative cells of cell types involved in a disease or disorder, todetermine if an antibody, a fusion protein, conjugated molecule, orcomposition of the present invention has a desired effect upon such celltypes. Preferably, the antibodies, the fusion proteins, the conjugatedmolecules, or compositions of the invention are also tested in in vitroassays and animal model systems prior to administration to humans.

Antibodies, fusion proteins, conjugated molecules, or compositions ofthe present invention for use in therapy can be tested for theirtoxicity in suitable animal model systems, including but not limited torats, mice, cows, monkeys, and rabbits. For in vivo testing for thetoxicity of an antibody, a fusion protein, a conjugated molecule, or acomposition, any animal model system known in the art may be used.

Efficacy in treating or preventing viral infection may be demonstratedby detecting the ability of an antibody, a fusion protein, a conjugatedmolecule, or a composition of the invention to inhibit the replicationof the virus, to inhibit transmission or prevent the virus fromestablishing itself in its host, or to prevent, ameliorate or alleviateone or more symptoms associated with viral infection. The treatment isconsidered therapeutic if there is, for example, a reduction is viralload, amelioration of one or more symptoms or a decrease in mortalityand/or morbidity following administration of an antibody, a fusionprotein, a conjugated molecule, or a composition of the invention.Antibodies, fusion proteins, conjugated molecules, or compositions ofthe invention can also be tested for their ability to inhibit viralreplication or reduce viral load in in vitro and in vivo assays.

Efficacy in treating or preventing bacterial infection may bedemonstrated by detecting the ability of an antibody, a fusion proteinor a composition of the invention to inhibit the bacterial replication,or to prevent, ameliorate or alleviate one or more symptoms associatedwith bacterial infection. The treatment is considered therapeutic ifthere is, for example, a reduction is bacterial numbers, amelioration ofone or more symptoms or a decrease in mortality and/or morbidityfollowing administration of an antibody, a fusion protein or acomposition of the invention.

Efficacy in treating cancer may be demonstrated by detecting the abilityof an antibody, a fusion protein, a conjugated molecule, or acomposition of the invention to inhibit or reduce the growth ormetastasis of cancerous cells or to ameliorate or alleviate one or moresymptoms associated with cancer. The treatment is considered therapeuticif there is, for example, a reduction in the growth or metastasis ofcancerous cells, amelioration of one or more symptoms associated withcancer, or a decrease in mortality and/or morbidity followingadministration of an antibody, a fusion protein, a conjugated molecule,or a composition of the invention. Antibodies, fusion proteins orcompositions of the invention can be tested for their ability to reducetumor formation in in vitro, ex vivo, and in vivo assays.

Efficacy in treating inflammatory disorders may be demonstrated bydetecting the ability of an antibody, a fusion protein, a conjugatedmolecule, or a composition of the invention to reduce or inhibit theinflammation in an animal or to ameliorate or alleviate one or moresymptoms associated with an inflammatory disorder. The treatment isconsidered therapeutic if there is, for example, a reduction is ininflammation or amelioration of one or more symptoms followingadministration of an antibody, a fusion proteins, a conjugated molecule,or a composition of the invention.

Antibodies, fusion proteins, conjugated molecules, or compositions ofthe invention can be tested in vitro and in vivo for the ability toinduce the expression of cytokines (e.g., IFN-α, IFN-β, IFN-γ, IL-2,IL-3, IL-4, IL-5, IL-6, IL10, IL-12, and IL-15) and activation markers(e.g., CD28, ICOS, and SLAM). Techniques known to those of skill in theart can be used to measure the level of expression of cytokines andactivation markers. For example, the level of expression of cytokinescan be measured by analyzing the level of RNA of cytokines by, forexample, RT-PCR and Northern blot analysis, and by analyzing the levelof cytokines by, for example, immunoprecipitation followed by Westernblot analysis or ELISA.

Antibodies, fusion proteins, conjugated molecules, or compositions ofthe invention can be tested in vitro and in vivo for their ability tomodulate the biological activity of immune cells, preferably humanimmune cells (e.g., T-cells, B-cells, and Natural Killer cells). Theability of an antibody, a fusion protein, a conjugated molecule, or acomposition of the invention to modulate the biological activity ofimmune cells can be assessed by detecting the expression of antigens,detecting the proliferation of immune cells, detecting the activation ofsignaling molecules, detecting the effector function of immune cells, ordetecting the differentiation of immune cells. Techniques known to thoseof skill in the art can be used for measuring these activities. Forexample, cellular proliferation can be assayed by 3H-thymidineincorporation assays and trypan blue cell counts. Antigen expression canbe assayed, for example, by immunoassays including, but are not limitedto, competitive and non-competitive assay systems using techniques suchas Western blots, immunohistochemistry, radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays and FACS analysis. The activationof signaling molecules can be assayed, for example, by kinase assays andelectrophoretic shift assays (EMSAs).

Antibodies, fusion proteins, conjugated molecules, or compositions ofthe invention can also be tested for their ability to increase thesurvival period of animals, preferably mammals and most preferablyhumans, suffering from a disease, disorder, or infection by at least25%, preferably at least 50%, at least 60%, at least 75%, at least 85%,at least 95%, or at least 99%. Further, antibodies, fusion proteins,conjugated molecules, or compositions of the invention can be tested fortheir ability reduce the hospitalization period of animals, preferablymammals and most preferably humans, suffering from a disease, disorder,or infection by at least 60%, preferably at least 75%, at least 85%, atleast 95%, or at least 99%. Techniques known to those of skill in theart can be used to analyze the function of the antibodies orcompositions of the invention in vivo.

5.9 Diagnostic Uses of Antibodies and Fusion Proteins

Labeled antibodies, fusion proteins, and conjugated molecules of theinvention can be used for diagnostic purposes to detect, diagnose, ormonitor diseases, disorders or infections. The invention provides forthe detection or diagnosis of a disease, disorder or infection,comprising: (a) assaying the expression of an antigen in cells or atissue sample of a subject using one or more antibodies thatimmunospecifically bind to the antigen; and (b) comparing the level ofthe antigen with a control level, e.g., levels in normal tissue samples,whereby an increase in the assayed level of antigen compared to thecontrol level of the antigen is indicative of the disease, disorder orinfection. The invention also provides for the detection or diagnosis ofa disease, disorder or infection, comprising (a) assaying the expressionof an antigen in cells or a tissue sample of a subject using one orfusion proteins or conjugated molecules of the invention that bind tothe antigen; and (b) comparing the level of the antigen with a controllevel, e.g., levels in normal tissue samples, whereby an increase ofantigen compared to the control level of the antigen is indicative ofthe disease, disorder or infection. Accordingly, the fusion protein orconjugated molecule comprises a bioactive molecule such as a ligand,cytokine or growth factor and the hinge-Fc region or fragments thereof,wherein the fusion protein or conjugated molecule is capable of bindingto an antigen being detected.

Antibodies of the invention can be used to assay antigen levels in abiological sample using classical immunohistological methods asdescribed herein or as known to those of skill in the art (e.g., seeJalkanen et al., J. Cell. Biol., 101:976-985, 1985; Jalkanen et al., J.Cell. Biol., 105:3087-3096, 1987). Other antibody-based methods usefulfor detecting protein gene expression include immunoassays, such as theenzyme linked immunosorbent assay (ELISA) and the radioimmunoassay(RIA). Suitable antibody assay labels are known in the art and includeenzyme labels, such as, alkaline phosphatase, glucose oxidase;radioisotopes, such as iodine (¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹²¹In), and technetium (^(99m)Tc); luminescentlabels, such as luminol; and fluorescent labels, such as fluorescein andrhodamine.

Fusion proteins can be used to assay antigen levels in a biologicalsample using, for example, SDS-PAGE and immunoassays known to those ofskill in the art.

One aspect of the invention is the detection and diagnosis of a disease,disorder, or infection in a human. In one embodiment, diagnosiscomprises: a) administering (for example, parenterally, subcutaneously,or intraperitoneally) to a subject an effective amount of a labeledantibody that immunospecifically binds to an antigen; b) waiting for atime interval following the administration for permitting the labeledantibody to preferentially concentrate at sites in the subject where theantigen is expressed (and for unbound labeled molecule to be cleared tobackground level); c) determining background level; and d) detecting thelabeled antibody in the subject, such that detection of labeled antibodyabove the background level indicates that the subject has the disease,disorder, or infection. In accordance with this embodiment, the antibodyis labeled with an imaging moiety which is detectable using an imagingsystem known to one of skill in the art. Background level can bedetermined by various methods including, comparing the amount of labeledmolecule detected to a standard value previously determined for aparticular system.

In another embodiment, diagnosis comprises: a) administering (forexample, parenterally, subcutaneously, or intraperitoneally) to asubject an effective amount of a labeled fusion protein or conjugatedmolecule that binds to an antigen or some other molecule; b) waiting fora time interval following the administration for permitting the labeledfusion protein or conjugated molecule to preferentially concentrate atsites in the subject where the antigen or other molecule is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled fusionprotein or conjugated molecule in the subject, such that detection oflabeled fusion protein above the background level indicates that thesubject has the disease, disorder, or infection. In accordance with thisembodiment, the fusion protein or conjugated molecule comprises abioactive molecule such as a ligand, cytokine or growth factor and ahinge-Fc region or a fragment thereof, wherein said fusion protein orconjugated molecule is labeled with an imaging moiety and is capable ofbinding to the antigen being detected.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of ^(99m)Tc. The labeled antibodywill then preferentially accumulate at the location of cells whichcontain the specific protein. In vivo tumor imaging is described in S.W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodiesand Their Fragments,” Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., MassonPublishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In one embodiment, monitoring of a disease, disorder or infection iscarried out by repeating the method for diagnosing the disease, disorderor infection, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the subject usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patient using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

5.10 Kits

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody, fusion protein,or conjugated molecule, of the invention, preferably in a purified form,in one or more containers. In a specific embodiment, the kits of thepresent invention contain a substantially isolated antigen as a control.Preferably, the kits of the present invention further comprise a controlantibody, fusion protein, or conjugated molecule which does not reactwith the antigen included in the kit. In another specific embodiment,the kits of the present invention contain a means for detecting thebinding of an antibody, fusion protein, or conjugated molecule, to anantigen (e.g., the antibody, fusion protein, or conjugated molecule, maybe conjugated to a detectable substrate such as a fluorescent compound,an enzymatic substrate, a radioactive compound or a luminescentcompound, or a second antibody which recognizes the first antibody maybe conjugated to a detectable substrate). In specific embodiments, thekit may include a recombinantly produced or chemically synthesizedantigen. The antigen provided in the kit may also be attached to a solidsupport. In a more specific embodiment the detecting means of theabove-described kit includes a solid support to which antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to theantigen can be detected by binding of the said reporter-labeledantibody.

5.11 In Vitro and In Vivo Assays for Extended Half-Life of Modified IgGHinge-Fc Fragments

The binding ability of modified IgGs and molecules comprising an IgGconstant domain of FcRn fragment thereof to FcRn can be characterized byvarious in vitro assays. PCT publication WO 97/34631 by Ward disclosesvarious methods in detail and is incorporated herein in its entirety byreference.

For example, in order to compare the ability of the modified IgG orfragments thereof to bind to FcRn with that of the wild type IgG, themodified IgG or fragments thereof and the wild type IgG can beradio-labeled and reacted with FcRn-expressing cells in vitro. Theradioactivity of the cell-bound fractions can be then counted andcompared. The cells expressing FcRn to be used for this assay arepreferably endothelial cell lines including mouse pulmonary capillaryendothelial cells (B10, D2.PCE) derived from lungs of B10.DBA/2 mice andSV40 transformed endothelial cells (SVEC) (Kim et al., J. Immunol.,40:457-465, 1994) derived from C3H/HeJ mice. However, other types ofcells, such as intestinal brush borders isolated from 10- to 14-day oldsuckling mice, which express sufficient number of FcRn can be also used.Alternatively, mammalian cells which express recombinant FcRn of aspecies of choice can be also utilized. After counting the radioactivityof the bound fraction of modified IgG or that of wild type, the boundmolecules can be then extracted with the detergent, and the percentrelease per unit number of cells can be calculated and compared.

Affinity of modified IgGs for FcRn can be measured by surface plasmonresonance (SPR) measurement using, for example, a BIAcore 2000 (BIAcoreInc.) as described previously (Popov et al., Mol. Immunol., 33:493-502,1996; Karlsson et al., J. Immunol. Methods, 145:229-240, 1991, both ofwhich are incorporated by reference in their entireties). In thismethod, FcRn molecules are coupled to a BIAcore sensor chip (e.g., CM5chip by Pharmacia) and the binding of modified IgG to the immobilizedFcRn is measured at a certain flow rate to obtain sensorgrams using BIAevaluation 2.1 software, based on which on- and off-rates of themodified IgG, constant domains, or fragments thereof, to FcRn can becalculated.

Relative affinities of modified IgGs or fragments thereof, and the wildtype IgG for FcRn can be also measured by a simple competition bindingassay. Unlabeled modified IgG or wild type IgG is added in differentamounts to the wells of a 96-well plate in which FcRn is immobilize. Aconstant amount of radio-labeled wild type IgG is then added to eachwell. Percent radioactivity of the bound fraction is plotted against theamount of unlabeled modified IgG or wild type IgG and the relativeaffinity of the modified hinge-Fc can be calculated from the slope ofthe curve.

Furthermore, affinities of modified IgGs or fragments thereof, and thewild type IgG for FcRn can be also measured by a saturation study andthe Scatchard analysis.

Transfer of modified IgG or fragments thereof across the cell by FcRncan be measured by in vitro transfer assay using radiolabeled IgG orfragments thereof and FcRn-expressing cells and comparing theradioactivity of the one side of the cell monolayer with that of theother side. Alternatively, such transfer can be measured in vivo byfeeding 10- to 14-day old suckling mice with radiolabeled, modified IgGand periodically counting the radioactivity in blood samples whichindicates the transfer of the IgG through the intestine to thecirculation (or any other target tissue, e.g., the lungs). To test thedose-dependent inhibition of the IgG transfer through the gut, a mixtureof radiolabeled and unlabeled IgG at certain ratio is given to the miceand the radioactivity of the plasma can be periodically measured (Kim etal., Eur. J. Immunol., 24:2429-2434, 1994).

The half-life of modified IgG or fragments thereof can be measure bypharmacokinetic studies according to the method described by Kim et al.(Eur. J. of Immuno. 24:542, 1994), which is incorporated by referenceherein in its entirety. According to this method, radiolabeled modifiedIgG or fragments thereof is injected intravenously into mice and itsplasma concentration is periodically measured as a function of time, forexample, at 3 minutes to 72 hours after the injection. The clearancecurve thus obtained should be biphasic, that is, α-phase and β-phase.For the determination of the in vivo half-life of the modified IgGs orfragments thereof, the clearance rate in β-phase is calculated andcompared with that of the wild type IgG.

6. EXAMPLES

The following examples illustrate the production, isolation, andcharacterization of modified hinge-Fc fragments that have longer in vivohalf-lives.

6.1 Library Construction

6.1.1 Reagents

All chemicals were of analytical grade. Restriction enzymes andDNA-modifying enzymes were purchased from New England Biolabs, Inc.(Beverly, Mass.). Oligonucleotides were synthesized by MWG Biotech, Inc.(High Point, N.C.). pCANTAB5E phagemid vector, anti-E-tag-horseradishperoxydase conjugate, TG1 E. Coli strain, IgG Sepharose 6 Fast Flow andHiTrap protein A columns were purchased from APBiotech, Inc.(Piscataway, N.J.). VCSM13 helper phage and the Quick change mutagenesiskit were obtained from Stratagene (La Jolla, Calif.). CJ236 E. colistrain was purchased from Bio-Rad (Richmond, Calif.). BCA Protein AssayReagent Kit was obtained from Pierce (Rockford, Ill.). Lipofectamine2000 was purchased from Invitrogen, Inc. (Carlsbad, Calif.).

6.1.2 Expression and Purification of Murine and Human FcRn

The amino acid sequences of human and mouse FcRn are SEQ ID NOs. 84 and85, respectively (see also Firan et al., Intern. Immunol., 13:993-1002,2001 and Popov et al., Mol. Immunol., 33:521-530, 1996, both of whichare incorporated herein by reference in their entireties). Human FcRnwas also obtained following isolation from human placenta cDNA(Clontech, Palo Alto, Calif.) of the genes for human β2-microglobulin(Kabat et al., 1991, Sequences of Proteins of Immunological Interest,U.S. Public Health Service, National Institutes of Health, Washington,D.C.) and codons −23 to 267 of the human a chain (Story et al., J. Exp.Med., 180:2377-2381, 1994) using standard PCR protocols. Light and heavychains along with their native signal sequence (Kabat et al., 1991,supra; Story et al., supra) were cloned in pFastBac DUAL and pFastBac1bacmids, respectively, and viral stocks produced in Spodopterafrugiperda cells (Sf9) according to the manufacturer's instructions(Invitrogen, Carlsbad, Calif.). High-Five cells were infected at amultiplicity of infection of 3 with the baculoviruses encoding α and β2chains using commercially available protocols (Invitrogen). Recombinanthuman FcRn was purified as follows: supernatant of infected insect cellswas dialyzed into 50 mM MES (2-N-[Morpholino]ethansulfonic acid) pH 6.0and applied to a 10 ml human IgG Sepharose 6 Fast Flow column(APBiotech, Piscataway, N.J.). Resin was washed with 200 ml 50 mM MES pH6.0 and FcRn eluted with 0.1 M Tris-Cl pH 8.0. Purified FcRn wasdialyzed against 50 mM MES pH 6.0, flash frozen and stored at −70° C.The purity of proteins was checked by SDS-PAGE and HPLC.

6.1.3 Preparation of TAA-Containing ssDNA Uracil Template

Construction of the libraries was based on a site directed mutagenesisstrategy derived from the Kunkel method (Kunkel et al., Methods Enzymol.154:367-382, 1987). A human hinge-Fc gene spanning amino acid residues226-478 (Kabat numbering, Kabat et al., 1991, supra) derived fromMEDI-493 human IgG1 (Johnson et al., J. Infect. Disease, 176:1215-1224,1997), was cloned into the pCANTAB5E phagemid vector as an SfiI/NotIfragment. Four libraries were generated by introducing random mutationsat positions 251, 252, 254, 255, 256 (library 1), 308, 309, 311, 312,314 (library 2), 385, 386, 387, 389 (library 3) and 428, 433, 434, 436(library 4). Briefly, four distinct hinge-Fc templates were generatedusing PCR by overlap extension (Ho et al., Gene, 15:51-59, 1989), eachcontaining one TAA stop codon at position 252 (library 1), 310 (library2), 384 (library 3) or 429 (library 4), so that only mutagenizedphagemids will give rise to Fc-displaying phage.

Each TAA-containing single-stranded DNA (TAAssDNA) was then prepared asfollows: a single CJ236 E. coli colony harboring one of the fourrelevant TAA-containing phagemids was grown in 10 ml 2×YT mediumsupplemented with 10 μg/ml chloramphenicol and 100 μg/ml ampicillin. At0D₆₀₀=1, VCSM13 helper phage was added to a final concentration of 10¹⁰pfu/ml. After 2 hours, the culture was transferred to 500 ml of 2×YTmedium supplemented with 0.25 μg/ml uridine, 10 μg/ml chloramphenicol,30 μg/ml kanamycin, 100 μg/ml ampicillin and grown overnight at 37° C.Phage were precipitated with PEG6000 using standard protocols (Sambrooket al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring HarborPress, Cold Spring Harbor, N.Y., Vols. 1-3) and purified using theQiaprep Spin M13 Kit (Qiagen, Valencia, Calif.) according to themanufacturer's instructions. 10 to 30 μg of each uracil-containingTAAssDNA template was then combined with 0.6 μg of the followingphosphorylated oligonucleotides (randomized regions underlined) in 50 mMTris-HCl, 10 mM MgCl₂, pH 7.5 in a final volume of 250 μl:

Library 1: (SEQ ID NO: 120)5′-CATGTGACCTCAGGSNNSNNSNNGATSNNSNNGGTGTCCTTGGGTTT TGGGGGG-3′ Library 2:(SEQ ID NO: 121) 5′-GCACTTGTACTCCTTGCCATTSNNCCASNNSNNGTGSNNSNNGGTGAGGACGC-3′ Library 3: (SEQ ID NO: 122)5′-GGTCTTGTAGTTSNNCTCSNNSNNSNNATTGCTCTCCC-3′ Library 4: (SEQ ID NO: 123)5′-GGCTCTTCTGCGTSNNGTGSNNSNNCAGAGCCTCATGSNNCACGGAG CATGAG-3′ where N= A, C, T or G and S = G or C.

6.1.4 Synthesis of Heteroduplex DNA

Appropriate, degenerate oligonucleotides were phosphorylated in thepresence of T4 polynucleotide kinase using the standard protocol. Ten to30 μg of ssDNA U template and 0.6 μg of phosphorylated oligonucleotidewere combined in 50 mM Tris-HCl containing 10 mM MgCl₂, pH 7.5, to afinal volume of 250 μl and incubated at 90° C. for 2 minutes, 50° C. for3 minutes, and 20° C. for 5 minutes. Synthesis of the heteroduplex DNAwas carried out by adding 30 units of both T4 DNA ligase and T7 DNApolymerase in the presence of 0.4 mM ATP, 1 mM dNTPs and 6 mM DTT andthe mixture was incubated for 4 hours at 20° C. The heteroduplex DNAthus produced was then purified and desalted using Qiagen Qiaquick DNApurification Kit (Qiagen, CA).

6.1.5 Electroporation

300 μl electrocompetent TG1 E. coli cells were electroporated with 1 to5 μg of the heteroduplex DNA in a 2.5 kV field using 200 S2 and 25 μFcapacitance until a library size of 1×10⁸ (library 1 and 2) or 1×10⁷(library 3 and 4) was reached. The cells were resuspended in 2 ml SOCmedium and the procedure was repeated 6 to 10 times. The diversity wasassessed by titration of recombinant E. coli. The pulsed cells wereincubated in 50 ml SOC medium for 30 minutes at 37° C. under agitation,centrifuged, and resuspended in 500 ml 2×YT containing 100 μg/mlampicillin and 10¹⁰ pfu/ml of VCSM13 helper phage. The culture wasincubated overnight at 37° C. and the cells were pelleted bycentrifugation. The phage in the supernatant which express mutatedhinge-Fc portion on its GIII-coat protein were precipitated with PEG6000as previously described (Sambrook et al., 1989, supra) and resuspendedin 5 ml of 20 mM MES, pH 6.0.

6.2 Panning of the Library

Phage were panned using an ELISA-based approach. A 96-well ELISA platewas coated with 100 μl/well of 0.01 mg/ml murine FcRn in sodiumcarbonate buffer, pH 9.0, at 4° C. overnight and then blocked with 4%skimmed milk at 37° C. for 2 hours. In each well of the coated plate,100-150 μl of the phage suspension (about 10¹³ phage in total) in 20 mMMES, pH 6.0, containing 5% milk and 0.05% Tween 20, were placed andincubated at 37° C. for two to three hours with agitation.

After the incubation, the wells were washed with 20 mM MES, pH 6.0,containing 0.2% Tween 20 and 0.3 M NaCl about thirty times at roomtemperature. The bound phage were eluted with 100 μl/well of PBS, pH7.4, at 37° C. for 30 minutes.

The eluted phage were then added to the culture of exponentially growingE. coli cells and propagation was carried out overnight at 37° C. in 250ml 2xYT supplemented with 100 μg/ml ampicillin and 10¹⁰ pfu/ml of VCSM13helper phage. Propagated phage were collected by centrifugation followedby precipitation with PEG and the panning process was repeated up to atotal of six times.

For the phage library containing mutations in residues 308-314 (H310 andW313 fixed), the phage expressing hinge-Fc region with higher affinitiesfor FcRn were enriched by each panning process as shown in Table IV. Thepanning results of the library for the mutations in the residues 251-256(1253 fixed) and that of the library for the mutations in the residues428-436 (H429, E430, A431, L432, and H435 fixed), are shown in Tables Vand VI, respectively. Furthermore, the panning results of the libraryfor the mutations in the residues 385-389 (E388 fixed) is shown in TableVII.

TABLE IV PANNING OF LIBRARY (RESIDUES 308-314; H310 AND W313 FIXED)pCANTAB5E-KUNKEL-muFcRn (MURINE FcRn) OUTPUT ENRICHMENT PANNING +FcRn−FcRn RATIO 1st Round 1.1 × 10⁵   0.5 × 10⁵ 2 2nd Round 1 × 10⁴ 0.2 ×10⁴ 5 3rd Round 9 × 10⁴ 0.3 × 10⁴ 30 4th Round 3 × 10⁵   2 × 10⁴ 15

TABLE V PANNING OF LIBRARY (RESIDUES 251-256; I253 FIXED)pCANTAB5E-KUNKEL-muFcRn OUTPUT ENRICHMENT PANNING +FcRn −FcRn RATIO 1stRound 2.5 × 10⁵ 1 × 10⁵ 2.5 2nd Round   6 × 10⁴ 2 × 10⁴ 3.0 3rd Round  8 × 10⁵ 4 × 10⁴ 20 4th Round 1.2 × 10⁶ 5 × 10⁴ 24 5th Round 3.0 × 10⁶6 × 10⁴ 50

TABLE VI PANNING OF LIBRARY (RESIDUES 428-436; H429, E430, A431, L432,AND H435 FIXED) pCANTAB5E-KUNKEL-muFcRn OUTPUT ENRICHMENT PANNING +FcRn−FcRn RATIO 1st Round 2.3 × 10⁵   0.9 × 10⁵   2.5 2nd Round 3 × 10⁴ 1 ×10⁴ 3 3rd Round 2 × 10⁵ 2 × 10⁴ 10 4th Round 8 × 10⁵ 5 × 10⁴ 16

TABLE VII PANNING OF LIBRARY (RESIDUES 385-389; E388 FIXED)pCANTAB5E-KUNKEL-muFcRn OUTPUT ENRICHMENT PANNING +FcRn −FcRn RATIO 1stRound 4.2 × 10⁵ 3.8 × 10⁵   1.1 2nd Round   5 × 10⁴ 0.3 × 10⁴   17 3rdRound 3.5 × 10⁵ 1 × 10⁴ 35 4th Round 5.5 × 10⁵ 4 × 10⁴ 14 5th Round 7.5× 10⁵ 5 × 10⁴ 15 6th Round   2 × 10⁶ 1 × 10⁵ 20

6.3 Identification of Isolated Clones from Panning

After each panning process, phage were isolated and the nucleic acidsencoding the expressed peptides which bound to FcRn were sequenced by astandard sequencing method such as by dideoxynucleotide sequencing(Sanger et al., Proc. Natl. Acad. Sci. USA, 74:5463-5467, 1977) using aABI3000 genomic analyzer (Applied Biosystems, Foster City, Calif.).

As a result of panning, two mutants were isolated from the phage librarycontaining mutations in residues 308-314 (H310 and W313 fixed), thirteenmutants from the library for residues 251-256 (1253 fixed), six mutantsfrom the library for residues 428-436 (H429, E430, A431, L432, and H435fixed), and nine mutants from the library for residues 385-389 (E388fixed). The mutants isolated from the libraries are listed in TableVIII.

TABLE VIII MUTANTS ISOLATED BY PANNING LIBRARY MUTANTS* 251-256Leu Tyr Ile Thr Arg Glu (SEQ ID NO: 90) Leu

 Ile Ser Arg Thr (SEQ ID NO: 91) Leu

 Ile Ser Arg Ser (SEQ ID NO: 92) Leu

 Ile Ser Arg

(SEQ ID NO: 93) Leu

 Ile Ser Arg

(SEQ TD NO: 94) Leu

 Ile Ser Arg Thr (SEQ ID NO: 95) Leu Tyr Ile Ser Leu Gln (SEQ ID NO: 96)Leu Phe Ile Ser Arg Asp (SEQ ID NO: 97) Leu Phe Ile Ser Arg Thr (SEQ IDNO: 98) Leu Phe Ile Ser Arg Arg (SEQ ID NO: 99) Leu Phe Ile Thr Gly Ala(SEQ ID NO: 100) Leu Ser Ile Ser Arg Glu (SEQ ID NO: 101) Arg Thr IleSer Ile Ser (SEQ ID NO: 102) 308-314 Thr Pro  His Ser Asp Trp Leu (SEQID NO: 103) Ile Pro His Glu Asp Trp Leu (SEQ ID NO: 104) 385-389Arg Thr Arg Glu Pro (SEQ ID NO: 105)

 

 Pro Glu

(SEQ ID NO: 106) Ser Asp Pro Glu Pro (SEQ ID NO: 107) Thr Ser His GluAsn (SEQ ID NO: 108) Ser Lys Ser Glu Asn (SEQ ID NO: 109) His Arg SerGlu Asn (SEQ ID NO: 110) Lys Ile Arg Glu Asn (SEQ ID NO: 111) Gly IleThr Glu Ser (SEQ ID NO: 112) Ser Met Ala Glu Pro (SEQ ID NO: 113)428-436 Met His Glu Ala Leu

 

 His

(SEQ ID NO: 114) Met His Glu Ala Leu His Phe His His (SEQ ID NO: 115)Met His Glu Ala Leu Lys Phe His His (SEQ ID NO: 116) Met His Glu Ala LeuSer Tyr His Arg (SEQ ID NO: 117) Thr His Glu Ala Leu His Tyr His Thr(SEQ ID NO: 118) Met His Glu Ala Leu His Tyr His Tyr (SEQ ID NO: 119)*Substituting residues are indicated in bold faceThe underlined sequences in Table VIII correspond to sequences thatoccurred 10 to 20 times in the final round of panning and the sequencesin italics correspond to sequences that occurred 2 to 5 times in thefinal round of panning. Those sequences that are neither underlined noritalicized occurred once in the final round of panning.

6.4 Expression and Purification of Soluble Mutant Hinge-Fc Region

The genes encoding mutated hinge-Fc fragments are excised withappropriate restriction enzymes and recloned into an expression vector,for example, VβpelBhis (Ward, J. Mol. Biol., 224:885-890, 1992). Vectorscontaining any other type of tag sequence, such as c-myc tag,decapeptide tag (Huse et al., Science, 246:1275-1281, 1989), Flag™(Immunex) tags, can be used. Recombinant clones, such as E. coli, aregrown and induced to express soluble hinge-Fc fragments, which can beisolated from the culture media or cell lysate after osmotic shock,based on the tag used, or by any other purification methods well knownto those skilled in the art and characterized by the methods as listedbelow.

6.5 Construction, Production and Purification of IgG1 Variants

Representative Fc mutations such as 1253A, M252Y/S254T/T256E, M252W,M252Y, M252Y/T256Q, M252F/T256D, V308T/L309P/Q311S, G385D/Q386P/N389S,G385R/Q386T/P387R/N389P, H433K/N434F/Y436H, and N434F/Y436 wereincorporated into the human IgG1 MEDI-493 (SYNAGIS®) (Johnson et al.,1997, supra). The heavy chain was subjected to site-directed mutagenesisusing a Quick Change Mutagenesis kit (Stratagene, La Jolla, Calif.)according to the manufacturer's instructions and sequences were verifiedby didoxynucleotide sequencing using a ABI3000 (Applied Biosystems,Foster City, Calif.) sequencer. The different constructions wereexpressed transiently in human embryonic kidney 293 cells using a CMVimmediate-early promoter and dicistronic operon in which IgG1/V_(H) iscosecreted with IgG1/V_(L) (Johnson et al., 1997, supra). Transfectionwas carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.)and standard protocols. IgGs were purified from the conditioned mediadirectly on 1 ml HiTrap protein A columns according to themanufacterer's instructions (APBiotech).

6.6 Characterization of Mutated Hinge-Fc Region

6.6.1 In Vitro Characterization HPLC and SDS-PAGE

Following the purification, general characteristics such as molecularweight and bonding characteristics of the modified hinge-Fc fragmentsmay be studied by various methods well known to those skilled in theart, including SDS-PAGE and HPLC.

FcRn Binding Assay

Binding activity of modified hinge-Fc fragments can be measured byincubating radio-labeled wild-type hinge-Fc or modified hinge-Fc withthe cells expressing either mouse or human FcRn. Typically, endothelialcell lines such as SV40 transformed endothelial cells (SVEC) (Kim etal., J. Immunol., 40:457-465, 1994) are used. After incubation with thehinge-Fc fragments at 37° C. for 16-18 hours, the cells are washed withmedium and then detached by incubation with 5 mM Na₂EDTA in 50 mMphosphate buffer, pH 7.5, for 5 minutes. The radioactivity per 10⁷ cellsis measured.

Then, the cells are resuspended in 2 ml of 2.5 mg/ml CHAPS, 0.1 MTris-HCl pH 8.0 containing 0.3 mg/ml PMSF, 25 mg/ml pepstatin and 0.1mg/ml aprotinin and incubated for 30 minutes at room temperature. Thecell suspension is then centrifuged and the supernatant separated. Theradioactivity of the supernatant is measured and used to calculate theamount of the hinge-Fc fragments extracted per 10⁷ cells.

The K_(d) for the interaction of wild type human IgG1 with murine andhuman FcRn (269 and 2527 nM, respectively) agree well with the valuesdetermined by others (265 and 2350 nM, respectively, Firan et al., 2001,supra). The I253A mutation virtually abolishes binding to human andmurine FcRn, as reported by others (Kim et al., Eur. J. Immunol.,29:2819-2825, 1991; Shields et al., J. Biol. Chem., 276:6591-6604,2001). This is not the result of misfolding of the antibody as thismutant retains the same specific activity than the wild type molecule(SYNAGIS®) in a microneutralization assay (Johnson et al., 1997, supra;data not shown).

Human IgG1 mutants with increased binding affinity towards both murineand human FcRn were generated (Table VIII). Improvements in complexstability were overall less marked for the human IgG1-human FcRn pairthan for the human IgG1-murine FcRn compared to wild type IgG1 were30-(ΔΔG=2.0 kcal/mol for N434F/Y436H) and 11-(ΔΔG=1.4 kcal/mol forM252Y/S254Y/S254T/T256E) fold, respectively. However, ranking of themost critical positions remain unchanged when comparing human and murineFcRn: the largest increases in IgG1-murine FcRn complex stability(ΔΔG>1.3 kcal/mol) occurred on mutations at positions 252, 254, 256(M252Y/S254T/T256E and M252W) and 433, 434, 436 (H433K/N434F/Y436H andN434F/Y436H). Likewise, the same mutations were found to have the mostprofound impact on the IgG1-human FcRn interaction and also resulted inthe largest increases in complex stability (ΔΔG>1.0 kcal/mol).Substitutions at positions 308, 309, 311, 385, 386, 387 and 389 hadlittle or no effect on the stability of the complexes involving human ormurine FcRn (ΔΔG<0.5 kcal/mol). Residues at the center of the Fc-FcRncombining site contribute significantly more to improvement in complexstability than residues at the periphery (FIG. 9).

Efficient binding of human Fc to murine FcRn apparently requires thepresence of several wild type Fc residues. For example, leucine is veryconserved at 251, arginine at 255, aspartic acid at 310, leucine at 314and methionine at 428 (FIG. 6). Another specificity trend is observedwhen one considers positions 308, 309, and 311 where threonine, proline,and serine, respectively, are very strongly favored over thecorresponding wild type residues (FIG. 6). However, generation of thisstrong consensus sequences does not correlate with the magnitude ofincrease in affinity as V308T/L309P/Q311S binds less than 2-fold betterthan the wild type IgG1 to both human and murine FcRn (Table IX).

Increases in affinity can be strongly dependent upon residuesubstitution at one ‘hot spot’ position. For example, the singlemutation M252Y causes an increase in binding to murine FcRn by 9-fold,whereas additional mutations bring little (M252Y/S254T/T256E) or no(M252Y/T256Q) added benefit. The same trend is observed for the humanreceptor, although to a lesser extent. Indeed, M252Y/S254T/T256E shows amarked improvement of 2.5-fold in affinity compared to M252Y. Thisprobably reflects the differences between the binding site of human andmurine FcRn (West and Bjorkman, Biochemistry, 39:9698-9708, 2000).

Phage-derived IgG1 mutants exhibiting a significant increase in affinitytowards murine FcRn (ΔΔG>1.3 kcal/mol) also showed significant bindingactivity to the receptor at pH 7.2 when compared to wild type IgG1(FIGS. 8A-H). IgG1 mutants with moderate increase in affinity (ΔΔG<0.3kcal/mol) bound very poorly at pH 7.2 (data not shown). In contrast,IgG1 mutants with large (ΔΔG>1.0 kcal/mol) increase in affinity towardshuman FcRn exhibited only minimal binding at pH 7.4 when compared towild type IgG1 (FIGS. 8A-H).

TABLE IX DISSOCIATION CONSTANTS AND RELATIVE FREE ENERGY CHANGES FOR THEBINDING OF IgG1/FC MUTANTS TO MURINE AND HUMAN FcRn* DissociationDissociation Constant ΔΔG Constant ΔΔG Fc/Murine (kcal/ Fc/Human (kcal/MUTANT FcRn (nM) mol) FcRn (mM) mol) wild type 269 ± 1  2527 ± 117 I253ANB NA NB NA M252Y/S254T/T256E 27 ± 6 1.4 225 ± 10 1.4 M252W 30 ± 1 1.3408 ± 24 1.1 M252Y 41 ± 7 1.1 532 ± 37 0.9 M252Y/T256Q 39 ± 8 1.1  560 ±102 0.9 M252F/T256D 52 ± 9 1.0  933 ± 170 0.6 V308T/L309P/Q311S 153 ± 230.3 1964 ± 84  0.1 G385D/Q386P/N389S 187 ± 10 0.2 2164 ± 331 0.1G385R/Q386T/P387R/ 147 ± 24 0.4 1620 ± 61  0.3 N389P H433K/N434F/Y436H14 ± 2 1.8 399 ± 47 1.1 N434F/Y436H  9 ± 1 2.0 493 ± 7  1.0 *Affinitymeasurements were carried out by BIAcore as described above. Residuenumbering is according to EU (Kabat et al., 1991, supra). Differences infree energy changes are calculated as the differences between the Δgs ofwild type and mutant reactions (ΔΔG = ΔG_(wild type) − ΔG_(mutant)). NB,no binding. NA, not-applicable.

FcRn-Mediated Transfer Assay

This assay follows the protocol disclosed in PCT publication WO97/34631. Radiolabeled modified hinge-Fc fragments at variousconcentration (1 μg/ml-1 mg/ml) are added to the one side of thetranswell and the transfer of the fragments mediated by FcRn-expressingmonolayer of the cells can be quantitated by measuring the radioactivityon the other side of the transwell.

6.6.2 In Vivo Pharmacokinetic Study

In order to determine the half-life of the modified IgG hinge-Fc,modified hinge-Fc fragments are radiolabelled with ¹²⁵I (approximatespecific activity of 10⁷ cpm/m) and dissolved in saline (pH 7.2). Thesolution is injected intravenously into BALB/c mice (Harlan,Indianapolis, Ind.), which have been given NaI-containing waterpreviously to block the thyroid, in a volume not more than 150 μl andwith a radioactivity of 10×10⁶-50×10⁶ cpm. The mice are bled from theretro-orbital sinus at various time points, for example, at 3 minutes to72 hours after the injection, into heparinized capillary tubes and theplasma collected from each sample is counted for radioactivity.

To generate the data provided in FIG. 10, 10 animals were used for eachmolecule assayed with 2.5 μg of antibody injected per animal. Antibodyserum levels were determined using an anti-human IgG ELISA (FIG. 10).There seems to be an inverse correlation between affinity to mouse FcRnand persistence in serum. This might be due to the significant amount ofbinding of the mutants observed at pH 7.2, which leads to thesequestration (i.e., lack of release in the serum) of the molecules.Preliminary data (not shown) suggests increased transport of the mutantsto the lung. Additionally, since the mutants exhibit lower levels ofbinding to human FcRn than murine FcRn (see FIGS. 8A-H), antibody serumlevels are expected to be higher in primates and humans.

6.6.3 Surface Plasmon Resonance Analyses

The interaction of soluble murine and human FcRn with immobilized humanIgG1 variants was monitored by surface plasmon resonance detection usinga BIAcore 3000 instrument (Pharmacia Biosensor, Uppsala, Sweden). Noaggregated material which could interfere with affinity measurements(van der Merwe et al., EMBO J., 12:4945-4954, 1993; van der Merwe etal., Biochemistry, 33:10149-10160, 1994) was detected by gel filtration.Protein concentrations were calculated by the bicinchoninic acid (BCA)method for both human and murine FcRn or using the 1% extinctioncoefficient at 280 nm of 1.5 for IgG1 wild type and variants. The latterwere coupled to the dextran matrix of a CM5 sensor chip (PharmaciaBiosensor) using an Amine Coupling Kit as described (Johnson et al.,supra). The protein concentrations ranged from 3-5 μg/ml in 10 mM sodiumacetate, pH 5.0. The activation period was set for 7 minutes at a flowrate of 10 μl/min and the immobilization period was set to between 10and 20 minutes at a flow rate of 10 μl/min. Excess reactive esters werequenched by injection of 70 μl of 1.0 methanolamine hydrochloride, pH8.5. This typically resulted in the immobilization of between 500 and4000 resonance units (RU). Human and murine FcRn were buffer exchangedagainst 50 mM PBS buffer pH 6.0 containing 0.05% Tween 20. Dilutionswere made in the same buffer. All binding experiments were performed at25° C. with concentrations ranging from 120 to 1 μg/ml at a flow rate of5 to 10 μl/min; data were collected for 25 to 50 minutes and three1-minute pulses of PBS buffer pH 7.2 were used to regenerate thesurfaces. FcRn was also flowed over an uncoated cell and the sensorgramsfrom these blank runs subtracted from those obtained with IgG1-coupledchips. Runs were analyzed using the software BIAevaluation 3.1(Pharmacia). Association constants (K_(A)s) were determined fromScatchard analysis by measuring the concentration of free reactants andcomplex at equilibrium after correction for nonspecific binding. Inequilibrium binding BIAcore experiments (Karlsson et al., 1991, supra;van der Merwe et al., 1993, supra; van der Merwe et al., 1994, supra;Raghavan et al., Immunity, 1:303-315, 1994; Malchiodi et al., J. Exp.Med., 182:1833-1845, 1995), the concentration of the complex can beassessed directly as the steady-state response. The concentration offree analyte (human or murine FcRn) is equal to the bulk analyteconcentration since analyte is constantly replenished during sampleinjection. The concentration of free ligand on the surface of the sensorchip can be derived from the concentration of the complex and from thetotal binding capacity of the surface as K_(A)=R_(eq)/C(R_(max)−R_(eq))where C is the free analyte concentration, R_(eq) is the steady-stateresponse, and R_(max) is the total surface binding capacity.Rearranging, the equation reads: R_(eq)/C=K_(A)R_(max)−K_(A)R_(eq).

A plot of R_(eq)/C versus R_(eq) at different analyte concentrationsthus gives a straight line from which K_(A) can be calculated (see TableIX). Errors were estimated as the standard deviation for two or threeindependent determinations and were <20%.

Representative mutations identified after panning libraries 1 through 4(FIG. 6, Table VIII) were introduced into the Fc portion of a humanIgG1. Injection of different concentrations of human or murine FcRn overthe immobilized IgG1 variants gave concentration-dependent binding.Typical resonance profiles for equilibrium binding of the mutantM252Y/S254T/T256E to murine and human FcRn are shown in FIGS. 7A and B.To estimate apparent K_(A)s, concentrations of FcRn ranging from 120 to1 μg/ml were used. In all cases, equilibrium (or near-equilibrium)binding levels were reached within 50 minutes. To estimate the increasein RU resulting from the non specific effect of protein on the bulkrefractive index, binding of FcRn to an uncoated cell was measured andthe sensorgrams from these blank runs subtracted from those obtainedwith IgG1-coupled chips. The scatchard plots for the binding of themutant M252Y/S254T/T256E to murine and human FcRn are shown in FIGS. 7Cand D. The plots were all linear, and apparent K_(A)s were calculatedfrom the relevant slopes. Measurements were carried out in duplicate ortriplicate and confirmed that the immobilized IgGs retained theiroriginal binding activity.

Since there are two non-equivalent binding sites on mouse IgG1 formurine FcRn with affinities of <130 nM and 6 μM (Sanchez et al.,Biochemistry, 38:9471-9476, 1999; Schuck et al., Mol. Immunol.,36:1117-1125, 1999; Ghetie and Ward, Ann. Rev. Immunol., 18:739-766,2000), the receptor was used in solution to avoid avidity effects thatarise when IgG1 binds to immobilized FcRn. Consistent with this,systematically higher affinities are observed when FcRn, rather thanIgG, immobilized on the biosensor chip (Popov et al., 1996, supra;Vaughn and Bjorkman, Biochemistry, 36:9374-9380, 1997; Martin andBjorkman, Biochemistry, 38:12639-12647; West and Bjorkman, Biochemistry,39:9698-9708, 2000). Under our experimental BIAcore conditions, mainlyinteractions corresponding to the higher-affinity association (i.e.single liganded-recptor) are measured, according for the linearity ofthe scatchard plots (FIGS. 7C and D).

BIAcore analysis was also used to compare the affinity of wild type IgG1and IgG1 mutants. Phage-derived IgG1 mutants exhibiting a significantincrease in affinity towards murine FcRn at pH 6.0 (ΔΔG>1.0 kcal/mol)also shoed significant binding to the mouse receptor at pH 7.2 with SPRsignal_(pH7.4)/SPR signal_(pH6.0)>0.6 at saturation. IgG1 mutants withmoderate increase in affinity towards murine FcRn at pH 6.0 (ΔΔG<0.4kcal/mol) bound very poorly to the mouse receptor at pH 7.2. Incontrast, IgG1 mutants exhibiting large affinity increase towards humanFcRn at pH 6.0 (ΔΔG>1.0 kcal/mol) only showed minimal binding to thehuman receptor at pH 7.4 with SPR signal_(pH7.4)/SPR signal_(pH6.0)<0.15at saturation.

Those skilled in the art will recognize, or be able to ascertain usingno more routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

6.7 Sequence Listing

The present specification is being filed with a Sequence Listingentitled “Seqlisting 10271-235-999.txt,” which incorporated herein byreference in its entirety.

1.-86. (canceled)
 87. A modified IgG comprising an IgG constant domain,wherein the IgG constant domain comprises a human IgG CH3 domain inwhich there is an amino acid substitution at amino acid residues 433,434 and 436 relative to a corresponding IgG comprising a wild-type humanIgG CH3 domain, numbered according to the EU numbering index of Kabat,wherein the modified IgG has an increased half-life compared to thehalf-life of the corresponding IgG comprising the wild-type human IgGCH3 domain, and wherein the substitution at amino acid residue 433 is asubstitution with a lysine, the substitution at amino acid residue 434is a substitution with a phenylalanine, and the substitution at aminoacid residue 436 is a substitution with a histidine.
 88. The modifiedIgG of claim 87, wherein the IgG constant domain is a human IgG constantdomain.
 89. The modified IgG according to claim 88, further comprisingone or more amino acid substitutions relative to the correspondingwild-type human IgG constant domain at one or more of amino acidresidues 251, 253, 255, 285-290, 308-314, 385-389, 429-433, and 435,numbered according to the Kabat EU numbering index.
 90. The modified IgGaccording to claim 89, wherein: the amino acid substitution at aminoacid residue 251 is a substitution with arginine; the amino acidsubstitution at amino acid residue 255 is a substitution with leucine,glycine or isoleucine; the amino acid substitution at amino acid residue308 is a substitution with a threonine or isoleucine; the amino acidsubstitution at amino acid residue 309 is a substitution with proline;the amino acid substitution at amino acid residue 311 is a substitutionwith serine, glutamic acid or leucine; the amino acid substitution atamino acid residue 312 is a substitution with alanine; the amino acidsubstitution at amino acid residue 314 is a substitution with alanine;the amino acid substitution at amino acid residue 385 is a substitutionwith arginine, aspartic acid, serine, threonine, histidine, lysine oralanine; the amino acid substitution at amino acid residue 386 is asubstitution with threonine, proline, aspartic acid, serine, lysine,arginine, isoleucine or methionine; the amino acid substitution at aminoacid residue 387 is a substitution with arginine, histidine, serine,threonine or alanine; the amino acid substitution at amino acid residue389 is a substitution with proline, serine or arginine; and the aminoacid substitution at amino acid residue 433 is a substitution withlysine, arginine, serine, isoleucine, proline or glutamine.
 91. Themodified IgG of claim 88, further comprising one or more amino acidsubstitutions relative to the corresponding wild-type human IgG constantdomain at amino acid residues 252, 254 or 256, numbered according to theKabat EU numbering index.
 92. The modified IgG of claim 91, wherein: theamino acid substitution at amino acid residue 252 is a substitution withtyrosine, phenylalanine, serine, tryptophan or threonine; the amino acidsubstitution at amino acid residue 254 is a substitution with threonine,and the amino acid substitution at amino acid residue 256 is asubstitution with serine, glutamic acid, arginine, glutamine, asparticacid, alanine or asparagine.
 93. The modified IgG of claim 88, furthercomprising an amino acid substitution relative to the correspondingwild-type human IgG constant domain at amino acid residue 314, numberedaccording to the Kabat EU numbering system.
 94. The modified IgGaccording to claim 93, wherein the amino acid substitution at amino acidresidue 314 is an alanine.
 95. The modified IgG of claim 88, wherein thehuman IgG constant domain with the amino acid substitutions has a higheraffinity for FcRn than a wild-type human IgG constant domain thereof.96. The modified IgG of claim 95, wherein the human IgG constant domainwith amino acid substitutions has a higher affinity for FcRn than awild-type human IgG constant domain thereof at pH 6.0 than at pH 7.4.97. The modified IgG of claim 88, wherein the modified IgG is a modifiedhuman IgG or a humanized IgG.
 98. The modified IgG of claim 88, whereinthe human IgG constant domain is the constant domain of IgG₁, IgG₂, IgG₃or Iga₄.
 99. The modified IgG of claim 98, wherein the human IgGconstant domain is the constant domain of IgG₁.
 100. The modified IgG ofclaim 97, wherein the human IgG is IgG₁, IgG₂, IgG₃ or IgG₄.
 101. Themodified IgG of claim 87 or 88, which immunospecifically binds to arespiratory syncytial virus (RSV) antigen.
 102. The modified IgGaccording to claim 101, which comprises: (a) a heavy chain variabledomain and light chain variable domain of palivizumab (SEQ ID NOS.: 7and 8, respectively); (b) a variable heavy (VH) complementarilydetermining region (CDR)1, VH CDR2, VH CDR3, variable light (VL) CDR1,VL CDR2 and VL CDR3 of palivizumab (SEQ ID NOS.: 1-6, respectively); (c)a VH CDR1, VH CDR2. VH CDR3, VL CDR1, VL CDR2 and VL CDR3 ofA4B4L1FR-S28R (SEQ ID NOS.:10, 19, 20, 39, 5, and 6, respectively); (d)a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of AFFF (SEQID NOS.:10, 2, 12, 14, 15 and 16, respectively); (e) VH CDR1, VH CDR2,VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of p12f2 (SEQ ID NOS.:18, 19, 20,22, 23 and 6, respectively); (f) a VH CDR1, VH CDR2, VH CDR3, VL CDR1,VL CDR2 and VL CDR3 of p12f4 (SEQ ID NOS.:18, 25, 20, 22, 27 and 6,respectively); (g) a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VLCDR3 of p11d4 (SEQ ID NOS.:18, 25, 29, 31, 32 and 6, respectively); (h)a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of Ale109 (SEQID NOS.:10, 25, 29, 22, 35 and 6, respectively); (i) a VH CDR1, VH CDR2,VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of A12a6 (SEQ ID NOS.:10, 37, 20,39, 35 and 6, respectively); (j) a VH CDR1, VH CDR2, CDR3, VL CDR1, VLCDR2 and VL CDR3 of A13c4 (SEQ ID NOS.:10, 41, 20, 22, 43, and 6,respectively); (k) a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VLCDR3 of A17d4 (SEQ ID NOS.:10, 45, 20, 47, 43, and 6, respectively); (l)a VH CDR1. VH CDR2, CDR3, VL CDR1, VL CDR2 and VL CDR3 of A4B4 (SEQ IDNOS.:10, 19, 20, 39, 50, and 6, respectively); (m) a VH CDR1, VH CDR2,VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of A8C7 (SEQ ID NOS.:10, 45, 29,31, 53, and 6, respectively); (n) a VH CDR1. VH CDR2, VH CDR3, VL CDR1,VL CDR2 and VL CDR3 of 1X-493L1FR (SEQ ID NOS.:1, 2, 3, 14, 5 and 6,respectively); (o) a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VLCDR3 of 113-3F4 (SEQ ID NOS.:10, 2, 29, 14, 15 and 6, respectively); (p)a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of M3H9 (SEQID NOS.:10, 2, 29, 14, 57 and 6, respectively); (q) a VH CDR1, VH CDR2,VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of Y10H6 (SEQ ID NOS.:10, 2, 29,14, 59 and 6, respectively); (r) a VH CDR1, VH CDR2, VH CDR3, VL CDR1,VL CDR2 and VL CDR3 of DG (SEQ ID NOS.:10, 2, 79, 14, 15 and 6,respectively); (s) a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VLCDR3 of AFFF(1) (SEQ ID NOS.:10, 2, 12, 14, 15 and 61, respectively);(t) a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of 6H8(SEQ ID NOS.:10, 2, 79, 14, 63 and 6, respectively); (u) a VH CDR1, VHCDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of L1-7E5 (SEQ ID NOS.:10,2, 79, 39, 15 and 6, respectively); (v) a VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2 and VL CDR3 of L215B10 (SEQ ID NOS.:10, 2, 79, 14, 66 and6, respectively); (w) a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 andVL CDR3 of A13AH (SEQ ID NOS.:10, 19, 29, 31, 69 and 6, respectively);(x) a VH CDR1, CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of A1H5 (SEQID NOS.:10, 25, 29, 72, 73 and 6, respectively); (y) a VH CDR1, VH CDR2,VH CDR3, VL CDR1, VL CDR2 and VL CDR3 of A4B4(1) (SEQ ID NOS.:10, 19,20, 39, 75 and 6, respectively); or (z) a VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2 and VL CDR3 of A4B4-F52S (SEQ ID NOS.:10, 19, 20, 39, 77and 6, respectively).
 103. The modified IgG of claim 87 or 88, whereinthe modified IgG immunospecifically binds to HER2, tumor necrosisfactor-alpha (TNF-α), transforming growth factor-β (TGF-β),interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-8 (IL-8), CD2,CD3, CD4, CD11a, CD14, CD18, CD20, CD23, CD25, CD33, CD52, CD64, CD80,CD147, CD40 ligand (CD40L), vascular endothelial growth factor (VEGF),intracellular adhesion molecule-3 (ICAM-3), epithelial growth factorreceptor (EGER), α_(v)β₃ integrin, α₄β₇ integrin, human leukocyteantigen (HLA), complement factor 5 (C5), immunoglobulin E (IgE),glycoprotein II_(b)/III_(a) receptor, CA125, 17-IA cell surface antigen,Factor VII, GD3 epitope, human immunodeficiency glycoprotein 120 (HIVgp120), hepatitis B virus (HBV) or cytomegalovirus (CMV).
 104. Themodified IgG of claim 87 or 88 which is isolated.
 105. A kit comprisingthe modified IgG of claim 87 or 88, in a container, and instructions foruse.
 106. An antibody conjugate comprising the modified IgG of claim 87or 88, and a detectable substance or a therapeutic moiety.
 107. Anantibody conjugate comprising the modified IgG of claim 102 and adetectable substance or a therapeutic moiety.
 108. A kit comprising theantibody conjugate according to claim 106, in a container, andinstructions for use.
 109. A kit comprising the antibody conjugateaccording to claim 107, in a container, and instructions for use.
 110. Anucleic acid comprising a nucleotide sequence encoding the modified IgGof claim
 103. 111. A nucleic acid comprising a nucleotide sequenceencoding the human IgG constant domain as defined in claim
 88. 112. Thenucleic acid of claim 110 which is isolated.
 113. The nucleic acid ofclaim 111 which is isolated.
 114. A host cell comprising the nucleicacid according to claim
 110. 115. A host cell comprising the nucleicacid according to claim
 111. 116. A pharmaceutical compositioncomprising the modified IgG of claim 87 or 88, and a pharmaceuticallyacceptable carrier.
 117. A pharmaceutical composition comprising themodified IgG of claim 101, and a pharmaceutically acceptable carrier.118. A pharmaceutical composition comprising the modified IgG of claim102, and a pharmaceutically acceptable carrier.
 119. A pharmaceuticalcomposition comprising the modified IgG of claim 106, and apharmaceutically acceptable carrier.
 120. A pharmaceutical compositioncomprising the modified IgG of claim 107, and a pharmaceuticallyacceptable carrier.
 121. A method of preventing or treating a disease ordisorder, comprising administering to a subject the modified IgG ofclaim 87 or
 88. 122. A method of preventing a RSV infection, comprisingadministering to a subject the modified IgG of claim 101 in themanufacture of a medicament for preventing RSV infection in a humansubject.
 123. A method of preventing a RSV infection, comprisingadministering to a subject the modified IgG of claim 102 in themanufacture of a medicament for preventing RSV infection in a humansubject.
 124. A method of treating a RSV infection, comprisingadministering to a subject the modified IgG of claim 101 in themanufacture of a medicament for preventing RSV infection in a humansubject.
 125. A method of treating a RSV infection, comprisingadministering to a subject the modified IgG of claim 102 in themanufacture of a medicament for preventing RSV infection in a humansubject.
 126. A method for detecting a disease or disorder in vitro,comprising: (a) contacting the modified IgG of claim 87 or 88 with asample from a subject; and (b) comparing the level of the antigen towhich the modified IgG immunospecifically binds to a control level,wherein an increase in level of the antigen in the sample compared tothe control level is indicative of the disease or disorder.
 127. Amethod for detecting a RSV infection in vitro, comprising: (a)contacting the modified IgG of claim 101 with a sample from a humansubject; and (b) comparing the level of the RSV antigen to which themodified IgG immunospecifically binds to a control level, wherein anincrease in level of the RSV antigen in the sample compared to thecontrol level is indicative of a RSV infection.
 128. A method fordetecting a RSV infection in vitro, comprising: (a) contacting themodified IgG of claim 102 with a sample from a human subject; and (b)comparing the level of the RSV antigen to which the modified IgGimmunospecifically binds to a control level, wherein an increase inlevel of the RSV antigen in the sample compared to the control level isindicative of a RSV infection.
 129. A method for diagnosing a disorderor disease, comprising: (a) administering to a subject the modified IgGof claim 87 or 88; (b) waiting for a time interval following theadministration for permitting the modified IgG to preferentiallyconcentrate at sites in the subject where the antigen to which themodified IgG immunospecifically binds is expressed; (c) determiningbackground level; and (d) detecting the modified IgG in the subject,wherein detection of the modified IgG above the background levelindicates that the subject has the disease or disorder.
 130. A methodfor diagnosing a RSV infection, comprising: (a) administering to a humansubject the modified IgG of claim 101; (b) waiting for a time intervalfollowing the administration for permitting the modified IgG topreferentially concentrate at sites in the subject where the RSV antigento which the modified IgG immunospecifically binds is expressed; (c)determining background level; and (d) detecting the modified IgG in thesubject, wherein detection of the modified IgG above the backgroundlevel indicates that the subject has a RSV infection.
 131. A method fordiagnosing a RSV infection, comprising: (a) administering to a humansubject the modified IgG of claim 102; (b) waiting for a time intervalfollowing the administration for permitting the modified IgG topreferentially concentrate at sites in the subject where the RSV antigento which the modified IgG immunospecifically binds is expressed; (c)determining background level; and (d) detecting the modified IgG in thesubject, wherein detection of the modified IgG above the backgroundlevel indicates that the subject has a RSV infection.
 132. The method ofclaim 130, wherein the modified IgG is a labeled form of the modifiedIgG.
 133. The method of claim 1, wherein the modified IgG is a labeledform of the modified IgG.
 134. The method of claim 121, wherein thesubject is a human.
 135. The method of claim 126, wherein the subject isa human.